Myxobacteria: Multicellularity and Differentiation

Seeking a convenient method of collecting myxospores than harvesting fruiting bodies, Dorothy Powelson’s group had explored techniques for converting vegetative cells into myxospores in liquid culture. A more detailed description of the process followed, which demonstrated that glycerol-induced myxospores were able to germinate, were resistant to elevated temperature, UV irradiation, and sonication, and mimicked the sequence of morphological stages during the formation of fruiting body myxospores. While David Zusman pointed out some important differences between glycerol-induced and fruiting body myxospores, glycerol induction quickly became a favorite vehicle for comparing the properties and processes of vegetative cells and myxospores, albeit with careful qualifications. It was suggested that the patchy quality of the peptidoglycan and the changes during glycerol induction were causally related to the shape change during myxospore formation. Despite structural differences between fruiting body and glycerol-induced myxospores, the Tn5lac insertion mutation, Ω7536, simultaneously blocked the development of glycerol-induced spores as well as fruiting body spores as they changed their shape from rod to sphere. By 1976 it had already become clear that a defining feature of myxobacterial behavior was the pervasive tendency of cells to maintain a high cell density. When the animal encounters nutrients and feeds, the package of myxospores may be deposited in the organic matter. The spores would germinate together and instantly create a feeding swarm of myxobacteria. Those experiments would constitute no more than one step in the overall task of understanding the whole organism that is revealed in the M. xanthus genome.

Molecular biologists have made great strides in understanding the genetics and molecular mechanisms that underlie social behavior in Myxococcus xanthus and a few other species. This chapter describes research on general ecological and evolutionary issues with the myxobacteria such as their diversity and distribution, population structure, and issues relating to their predatory behavior. Complete reliance on studies of natural populations to inform the understanding of myxobacterial ecology and evolution would limit the range of questions that can be addressed. These approaches not only are enhancing one's understanding of the myxobacteria per se, but also have the potential to inform the understanding of ecology and evolution more generally. A section briefly summarizes research as well as newer cultivation and molecular studies that have advanced the understanding of the diversity of the myxobacteria, their habitats, and the structure of their natural populations. The chapter describes some laboratory studies addressing the effects of abiotic variables on ecologically relevant phenotypes and the interactions of myxobacteria with prey species and how such interactions affect predator evolution. In recent years, the range of environmental conditions that may support active growth of myxobacteria has been shown to be greater than expected.

Myxococcus xanthus is a rod-shaped, gram-negative soil bacterium that, when subjected to nutrient deprivation, undergoes a developmental process culminating in the formation of a multicellular fruiting body filled with spores. For the purposes of this chapter, early development can be defined as events occurring from initiation to the start of aggregation at approximately the first 6 h poststarvation. In the chapter, the following tenets are addressed: how individual M. xanthus cells recognize starvation; how these cells perceive population starvation; and how individual cells integrate this information to ultimately initiate fruiting body formation and cellular differentiation. In summary, the balance of SocE and CsgA proteins in the cell is critical for sustaining the developmental program past initiation and is just one example of the unique aspects of the stringent response in this organism. Based on the current data, the simplest model for nutrient sensing still focuses on the cell’s ability to utilize its translational capacity as an overall measurement of starvation. It should be noted that other members of the Group B signaling mutants remain unmapped and are yet to be extensively characterized. A section exclusively discusses the properties of the bsgA mutants.

The intercellular C-signal has a fundamental role in fruiting body morphogenesis in Myxococcus xanthus. In this chapter, our current understanding of how the C-signal acts at the molecular level to induce and coordinate events that are separated in time and space is discussed. The first evidence for intercellular signals important for fruiting body formation came from the isolation of a collection of mutants that displayed nonautonomous developmental defects. Importantly, aggregation, sporulation, and C-signal-dependent gene expression were induced earlier than in wild-type cells, whereas the rippling stage was completely skipped. Random transposon mutagenesis followed by screening for mutants with deficiencies in C-signal-dependent responses, isolation of extragenic suppressors of a csgA insertion mutant, proteomics, and biochemical analyses have been instrumental in the identification of proteins in this pathway. Several regulatory mechanisms help to restrict the activity of the pathway to starving cells. First, starvation induces the stringent response, which, in turn, induces csgA transcription and A-signal accumulation, which induces fruA transcription. Secondly, starvation induces mrpAB expression, and MrpAB induces mrpC transcription. Thirdly, by an unknown mechanism secretion of MXAN0206, the protease likely to cleave p25, is induced. Moreover, the establishment of cell-biology-based methods with green fluorescent protein (GFP) fusion proteins and immunofluorescence are likely to result in the detailed understanding of the spatial organization of M. xanthus cells during starvation.

Colonies of Myxococcus xanthus on the surface of agar spread outward, and genetic studies of motility began with the isolation of mutants having abnormal spreading patterns. Motile strains can produce slime from either end and simply switch the producing end when they reverse. Bipolar slime secretion suggested that by trying to move in both directions simultaneously, cells were unable to make progress in either. A movie of single cells reversing, made by Lars Jelsbak, shows that cells simply stop momentarily before moving off in the opposite direction. How C-signaling gives rise to two different reversal patterns is explained by the signal transduction circuit. Rosa Yu isolated and characterized many new null-motility mutants and from the mutant phenotypes was able to draw two general conclusions about A-motility. First, all of her mutant strains that retained some A-motility, including mglB mutants, produced slime only at one end of each cell. The second conclusion was that mglA and all other “nonmotile” mutants that she had detected were secreting slime from both ends. The author suggests that the rapid reversals of mglA mutants are not due to signals from the reversal generator but are a statistical consequence of active slime secretion from both ends. For example, pilA, which encodes the pilin monomer, is one of the most highly transcribed genes in M. xanthus. The mechanism of gliding reversal that is proposed in this chapter explains how Mgl is shared by two engines that have no protein molecules in common.

Myxococcus xanthus is one of the diverse bacteria that display gliding motility, involving two mechanisms, called adventurous (A) and social (S) gliding motility. The genetic and phenotypic analysis of mutants defective in gliding motility has shown that gliding motility is more complex than flagellum-dependent motility. The first molecular insights about the mechanisms of gliding motility were gleaned from the results of genetic studies performed by Hodgkin and Kaiser, who used chemical mutagens to generate mutations that affected gliding. The phenotypes of gliding mutants can be measured in terms of the differences in their velocities of gliding. The more detailed molecular genetic analysis of the two mechanisms of gliding motility in M. xanthus has been facilitated by the use of transposon mutagenesis. M. xanthus cells that have a functional A gliding system are able to move as isolated cells on a solid surface. The majority of mutations that abolish S motility affect the production of type IV pili (TFP), the exopolysaccharide (EPS) component of fibrils, or the lipopolysaccharide (LPS) moiety of O-antigen. The function of EPS in social motility has been elucidated by phenotypic analysis of mutants of M. xanthus lacking EPS, a secreted polymer comprised primarily of N-acetylglucosamine (GlcNAc) and glucosamine (GlcN). M. xanthus has a complex life cycle. In the presence of adequate nutrients, the cells undergo vegetative growth and divide, but when the cells are starved of nutrients, they aggregate and form fruiting bodies containing myxospores.

Myxococcus xanthus has attracted much scientific interest because of its complex life cycle and morphogenetic potential. Western blot analysis showed that vegetative cells are highly methylated (about 50% of FrzCD is methylated) but cells that are starved are relatively unmethylated. The methylation changes in FrzCD suggest that during aggregation, FrzCD senses a chemical(s) produced by other cells that promotes cell movements towards aggregation centers. S-motility in M. xanthus, like twitching motility in Pseudomonas aeruginosa, has been shown to involve the extension and retraction of type IV pili localized at the leading cell pole. The stimulatory mutations blocked both vegetative swarming and developmental aggregation. The inhibitory mutations blocked developmental aggregation at low cell density, but not at high cell density, suggesting that specific methylation sites may be required for sensing low concentrations of developmental signals. The different phenotypes of the mutants observed in this study suggest that differential methylation could provide a potential signal input to the Frz chemosensory pathway. FrzS was also studied by constructing in-frame deletion mutants that express stable cryptic proteins; these mutants showed loss of function and/or distinct localization defects. Specifically, in-frame deletions of the pseudoreceiver domain, the coiled-coil domain, and a motif located at the very C terminus of the protein each resulted in aberrant localization and loss of function defects.

The majority of the analyses have focused on two-component signal transduction (TCST) systems, even though these are likely to compose only a fraction of the total systems that have evolved to translate information derived from the environment. The majority of all work on chemotaxis systems up until the last decade focused on the control of flagellum-based motility. Myxococcus xanthus is therefore an ideal organism for analysis of chemosensory signal transduction systems in bacteria. There are two primary reasons for our investigation into the role of the Che6 chemosensory system. First, the gene order is consistent with that found in other organisms known to utilize TFP-based motility. Second, a mutant allele known to suppress the csgA mutant developmental defect was mapped to socD (suppressor of csgA), which we now know is cotranscribed as part of the che6 operon. As M. xanthus does not possess phycobilisomes, Cpc7 is not likely to function as a phycocyanobilin lyase per se but might function in another light-dependent adaptation process. In summary, the chemosensory systems found in M. xanthus appear to have evolved in order to regulate functions that need temporal control mechanisms. In that regard M. xanthus may represent a case study encompassing both extremes where several systems regulate motility and several systems regulate alternative functions. Analysis of chemosensory signal transduction systems in each model organism is an excellent example of the modular nature of signal transduction and the evolution of bacterial genomes.

This chapter focuses primarily on transcriptional regulation of developmental genes. While the identification of developmentally regulated Myxococcus xanthus genes continues, now on a comprehensive genome-wide scale with the use of DNA microarray expression profiling, an understanding of the cis-acting DNA elements and trans-acting proteins (RNA polymerase [RNAP] with particular sigma factors, activators, and repressors) has emerged for a handful of developmental genes. In prokaryotes, sigma factors of RNAP play a key role in the regulation of gene expression by recognizing specific promoters and initiating transcription. As in other bacteria with a large number of sigma factors, such as Streptomyces coelicolor, most of the expansion of the σ70 family in M. xanthus is due to members of the extracytoplasmic function (ECF) subfamily. Transcriptional activation, rather than relief from repression, appears to account for induction of most developmentally regulated M. xanthus genes studied so far, though some genes are subject to both positive and negative control. ActB is encoded by the second gene of an operon that regulates the level of CsgA, the C-signaling protein, during M. xanthus development. Several transcription factors key to the M. xanthus developmental process have been identified. Some of these, like MrpC and FruA, emerged from transposon mutagenesis screens. Others, like σB-E and σ54, were identified by cross-hybridization with other sigma factor genes.

Myxobacteria live in an ever-changing environment and therefore require mechanisms to couple perception of environmental change with appropriate behavioral responses. The evolutionary success of two-component system (TCS) signaling pathways apparently stems from their adaptability to the regulation of diverse physiological processes, and this feature is illustrated well by the known TCSs of myxobacteria. The recent availability of genome sequences of four myxobacteria has enabled a comparative genomic analysis of TCS genes in myxobacterial genomes. TCSs can be consistently grouped into particular subfamilies by applying several different assessment criteria, including gene organization, domain architecture, and phylogenetic relationships. The major families of TCSs are usually named after archetypal family members from Escherichia coli. Phosphoaspartate residues in response regulators can also be hydrolyzed by extrinsic phosphatases. Surprisingly, there are no homologues of the Rap, Spo0E, YisI, YnzD, or CheZ phosphatases encoded in the Myxococcus xanthus genome, suggesting that modulation of signal flow by regulated phosphoaspartate phosphatase activity is not generally adopted by the myxobacteria. With the sequencing of multiple myxobacterial genomes it has become possible to use comparative genomics to gain novel insights into the TCSs of myxobacteria. Genomes can be assessed for the presence or absence of specific TCS homologues, lineage-specific changes in TCS properties can be identified, and in some cases changes in gene organization can guide searches for partner proteins.

Protein Ser/Thr/Tyr kinases and protein phosphatases function as biological switches that turn on and off signal transduction pathways, where they participate by phosphorylation and dephosphorylation. Based on analyzing the recently complete Myxococcus xanthus genome sequence database, 102 genes that encode putative protein Ser/Thr kinase (PSTK) and 34 genes of putative protein phosphatases (PPs) have been identified. This chapter first describes PSTKs and then PPs in M. xanthus. Four pp genes that encode protein phosphatases in the phosphoprotein phosphatase (PPP) superfamily are located close to pstk genes. In fact, four major superfamilies of phosphatases exist: phosphoprotein phosphatases (PPP), Mn2+-or Mg2+-dependent protein phosphatases (PPM), conventional phosphotyrosine phosphatases (CPTP), and low-molecular-mass phosphotyrosine phosphatases (LMMPTP). Among the 34 PPs, only four seem to be forming operons with protein kinases: three are PPPs, and the fourth one belongs to the PPM superfamily. There are four genes in the M. xanthus genome containing the Pfam for PP2C protein phosphatases (PF00481), including Pph1. As the signature for this family of proteins is also found in proteins such as nucleotidases, sphingomyelin phosphodiesterases, and 2’-3’ cAMP phosphodiesterases, as well as nucleases, it is difficult to estimate the exact number of PPP-type phosphatases in M. xanthus. The physiological roles of the PSTK signaling systems in M. xanthus are beginning to be understood at the molecular level. Their roles appear to be similar to those of the protein Ser/Thr and Tyr kinases in eukaryotes, known to regulate diverse cellular functions by forming kinase cascades with scaffold and adapter proteins.

Myxobacteria frequently occur as brightly colored colonies and sporangioles, due to the presence of carotenoids and/or other pigments. This chapter provides an update on the considerable amount of new information that has since been acquired with respect to the inducing signals, their reception and transduction, and the transcriptional regulation of the structural genes involved in carotenoid biosynthesis in Myxococcus xanthus. The observation that the action spectra for photolysis and carotenogenesis in M. xanthus were similar and corresponded closely to the absorption spectrum of the iron-containing protoporphyrin IX, led to the proposal that this compound was the photosensitizer that linked the processes of photolysis and carotenogenesis. The carD gene was identified in a screen for Car- mutants among a large collection of strains bearing Tn5 insertions. Recent studies have identified a new factor, CarG (the product of the gene directly downstream of carD), which is required in every CarD-dependent process analyzed. The CarA operator design and the mechanism underlying the repression-antirepression switch of PB have been subjected to detailed molecular analysis. The most studied phenomenon in M. xanthus is undoubtedly its striking ability to form multicellular fruiting bodies on starvation, a process that has served as a prokaryotic model for the study of cell-cell interactions and cellular differentiation. Studies with M. xanthus continue to provide insights into the general principles underlying the complexity of the biosynthetic pathways and regulatory mechanisms involved in eubacterial carotenogenesis.

This chapter covers in detail the polysaccharide-containing components of the Myxococcus xanthus cell envelope including the peptidoglycan (PG), lipopolysaccharide (LPS), extracellular matrix (ECM), and capsular exopolysaccharide (EPS). First, the PG was associated with substantial amounts of glycine, serine, and glucose. Second, the vegetative cell wall PG was suggested to be discontinuous in that whole sacculi were not isolated and trypsin and sodium dodecyl sulfate were able to completely disassociate the PG. At least two factors regulate MBHA accumulation. First, mbhA transcription, which is σ-54 dependent, increases during development. Second, the stability of the mbhA mRNA is increased during development. Interestingly, all of the LPS mutants identified by the authors' two laboratories mapped to three loci: two LPS O-antigen loci and one LPS core locus. The carbohydrate composition of wild-type LPS consists of glucose, mannose, rhamnose, arabinose, xylose, galactosamine, glucosamine, KDO (2-keto-3-deoxyoctulosonic acid), and 3-O-methylpentose and 6-O-methylgalactosamine. Signal perception by the Dif pathway involves Tfp. First, Tfp was found to be required for EPS production in M. xanthus. Second, Dif proteins function downstream of Tfp as demonstrated by genetic epistasis tests. Finally, Tfp do not appear to function as either exogenous or endogenous signals for the Dif pathway. Preliminary biochemical studies indicated that M. xanthus EPS contains at least five monosaccharides: galactose, glucosamine, glucose, rhamnose, and xylose. Studies of M. xanthus pili focus on their involvement in S-motility.

The first portion of this chapter, entitled “Catabolic Pathways,” deals with the catabolism of amino acids and lipids, as they are the principal carbon and energy sources derived from prey bacteria. The second portion of the chapter, entitled “Anabolic Pathways,” highlights the synthesis of lipids because of their unusual chemical structures in myxobacteria, and also the spore-specific components trehalose and ether lipids. Most myxobacteria, including Myxococcus xanthus, can catabolize prey microorganisms. M. xanthus utilizes amino acids and lipids as carbon and energy sources, incorporates purines and pyrimidines via salvage pathways, and fails to utilize sugars. In most cases there is excellent agreement between the presence of a particular amino acid catabolic pathway and the ability of that amino acid to stimulate growth in defined and minimal media. Lipid oxidation has been demonstrated by 14C-labeling experiments in Myxococcus virescens and methyloleate feeding in M. xanthus. Fatty acids are usually degraded by β oxidation, where two carbon acetate units are sequentially removed from the carboxyl end of the fatty acid, also known as the Δ terminus, as opposed to the methyl end or ω terminus. Monosaccharides are used for exopolysaccharide, peptidoglycan, and lipopolysaccharide biosynthesis. In Escherichia coli, trehalose is degraded to glucose by a periplasmic trehalase; no homolog exists in M. xanthus.

In the past 30 years myxobacteria have been established as proficient producers of various secondary metabolites and are regarded today as one of the few important sources for microbial natural products besides actinomycetes and fungi. Epothilone from Sorangium cellulosum recently successfully finished phase III clinical trials as an anticancer agent, as it represents a paclitaxel mimetic. Interestingly, secondary metabolites from myxobacteria often include structural elements which are rarely found in other sources. From the biosynthetic point of view, most of the isolated compounds represent hybrids of polyketides (PKs) and structural elements derived from nonribosomally made peptides (NRPs), whereas pure PKs are rarely found. Several members of this mixed polyketide/peptide structure have been identified that show differences in length of the polyene chain, methylation pattern, and/or hydroxylation of the terminal asparagine moiety. A genetic system is the limiting factor for identification and manipulation of secondary metabolite gene clusters in many organisms, including myxobacteria. The powerful combination of advanced cloning techniques and an advantageous expression host (which is currently far from being optimized) will allow the production of several interesting myxobacterial secondary metabolites in the future. It might even allow the production of new compounds directly from environmental DNA samples, eliminating the time-consuming or sometimes impossible isolation of pure cultures of any kind of putative producer of natural products.

This chapter discusses and compares the genomic sequences of Myxococcus xanthus DK1622 (hereafter referred to as DK1622 or strain DK1622) and Stigmatella aurantiaca DW4/3-1 (hereafter referred to as DW4/3-1 or strain DW4/3-1), two related aerobic, fruiting-body-forming myxobacteria. The structure and complexity of the S. aurantiaca fruiting body are the primary characteristics distinguishing it from M. xanthus DK1622, as well as the production of the signaling pheromone stigmolone, which may be analogous to the M. xanthus DK1622 quorum-sensing A-signal. Having the genome sequence of both organisms will greatly facilitate research into these and other myxobacteria-specific phenotypic traits. The M. xanthus DK1622 genome, initially sequenced to 4.5 coverage by Monsanto, was completed at The Institute for Genomic Research by additional random Sequencing. Both auto- and manual annotation were performed on the closed genome. Unlike most other prokaryotes, the myxobacteria exhibit social behavior and multicellular development. Such complex behavior is correlated with their large genomes. Gliding motility has also been extensively studied in M. xanthus DK1622, which, rather than swimming, hunts by gliding over the soil surface as a coordinated aggregation of thousands of cells, an essential element of its complex lifestyle. Myxobacteria feed by lysing cells of other bacteria and yeasts. Bioactive compounds synthesized and secreted through secondary metabolite biosynthetic gene clusters may aid predation and inhibit competition. The control of development in M. xanthus and S. aurantiaca is likely to be very similar based on the presence of DW4/3-1 orthologs to the genes controlling development in strain DK1622.

Currently, Myxococcus xanthus is the only species of myxobacteria to mature into the postgenomic phase, and therefore, this chapter focuses on this organism. The chapter begins with a discussion with genomic screens using transposable elements, continues with the paralogous analysis of gene families, and concludes with whole-genome yeast two-hybrid (Y2H) assays. Availability of the M. xanthus genome sequence makes the paralogous characterization of gene families possible. Both of the findings presented in the chapter are significant because they represent how the application of a genome sequence can aid in the identification of genes involved in specific processes such as development and vegetative growth. All of the postgenomics methods presented in the chapter focus on using the genome sequence to facilitate the characterization of genes using experimental techniques. One of the ultimate goals of myxobacterial genomics is to determine the complete set of genetic networks that contribute to the complex life cycle of the myxobacteria. Making sense of these data will require the concerted effort of the whole community of those involved in myxobacterial research to pool resources and share data. Exploratory work using microarrays is currently under way in an effort to identify the temporal expression of genes under a host of different environmental conditions. This work has thus far yielded insight into the number of genes associated with transcriptional activators (TAs) under vegetative conditions.

The DNA-dependent RNA polymerase with the main σ factor bound has been purified from vegetative cells to homogeneity as analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis with the aim to characterize σ factors and development-specific promoters in vitro. sigB and sigC expression was analyzed by Western blotting and reverse transcription-PCR. Both σ factors were found to be expressed during fruiting body formation and indole-induced sporulation but not after heat shock. The antiserum raised against SigB in this study cross-reacted with at least one further alternative σ factor whose expression pattern is similar to that of SigB. The stress protein HspA (formerly SP21) is synthesized during fruiting body formation, artificially induced sporulation, heat shock, and anoxia. An hspA deletion mutant behaved like the wild type during vegetative growth, fruiting body formation, sporulation, and spore germination; the thermotolerance was also not affected in the mutant. Importantly, the secreted activity was higher during development, especially during spore development, which may reflect an implication in the maturation of the spore coat. Secondary metabolites which exert some biological effect affect mainly electron transport. Examples of such compounds are aurafuron, aurachin, myxalamid, myxochromide, myxothiazol, and stigmatellin. Structurally most of these compounds are polyketides, peptides, or terpenoids. The most prominent feature of Stigmatella aurantiaca is the formation of a complex and highly structured fruiting body. This complexity represents the specific challenge of the Stigmatella system and distinguishes Stigmatella from Myxococcus and Sorangium.

In 1937 Imshenetski and Solntseva isolated a new species of cellulose-degrading myxobacteria, which they called Sorangium cellulosum. 16S rRNA gene sequencing of nine isolates of Sorangium and their comparison with Polyangium cellulosum as the reference strain proved a close phylogenetic relationship (evolutionary distance, less than 3% on the nucleotide level). The use of single antibiotics or better combinations of several antibiotics that act on different targets may be helpful, because Sorangium species usually turn out to be multiresistant. Cells of the suborder “Cystobacterineae” on the one hand and “Sorangiineae” and “Nannocystineae” on the other hand can easily be distinguished because they differ in cell morphology, as can be detected using phase-contrast microscopy. Strains of Byssophaga cruenta, characterized by their intense blood-red color, and the very common S. cellulosum strains are the only myxobacteria which degrade crystalline cellulose and can use it as the sole carbon source. At the Gesellschaft für Biotechnologische Forschung (GBF) an isolation and screening program was initiated in the late 1970s to evaluate the potential of the different genera of myxobacteria as producers of secondary metabolites. A ddc gene product, which was previously found only in eukaryotes, has also been identified in Sorangium strains. As discussed in this chapter, the fascinating microorganisms of the genus Sorangium attract more and more attention, because they undergo a complex life cycle, possess the largest bacterial genomes known to date, and show a high potential as producers of biotechnologically important natural products.

This chapter describes about the isolation of Bdellovibrio and myxobacteria from similar soil environments, and the employment of an arsenal of hydrolytic enzymes to kill and digest other bacteria to provide for their own growth and division. It concentrates on the ecology and motility systems of Bdellovibrio, followed by an overview of the hydrolytic enzymes used in prey digestion. It is clear that the nonobligately symbiotic or parasitic, predatory bacteria like Bdellovibrio and Myxobacteria have large genomes akin to those of heterotrophs; thus, determining whether transfer of predatory gene islands is responsible for apparently quite diverse bacteria adapting to fit certain predatory niches is not trivial and can ultimately be answered only by full comparative analysis of multiple predatory genomes, although there is little evidence of recent horizontal gene transfer in the Bdellovibrio bacteriovorus HD100 genome. Gliding motility genes are implicated in myxococcal motility, but these systems seem to be not present in Bdellovibrio, or they were made redundant so long ago that the genes involved are no longer recognizable as such. Homology searches found no significant homologues of Myxococcus genes involved in gliding. Bdellovibrio preys upon only gram-negative bacteria, albeit a wide range of these, as it penetrates the outer layers and enters the periplasmic space of its prey. By far the largest group of hydrolases in both bacteria is proteases; this probably reflects the importance to predatory bacteria in breaking down prey proteins for uptake and consumption by the predator.

This chapter focuses on Bacillus subtilis multicellularity, emphasizing the two-cell differentiation process of endospore formation and attempting to note similarities to Myxococcus xanthus. While cell growth, division, motility, and chemotaxis clearly play roles in forming bioconvection patterns, complex colonies, and macrofibers, these multicellular phenomena have not yet been subjected to systematic genetic analysis. In contrast, recently discovered multicellular behaviors of biofilm formation and swarming motility are rapidly being elucidated by genetic and genomic approaches. The most studied and best understood multicellular behaviors of B. subtilis are the development of genetic competence (the ability to take up exogenous DNA) and sporulation. The chapter summarizes the understanding of how morphogenesis and intercellular signaling control the activity of cell-specific s factors, focusing on recent progress and attempting to identify questions that remain. It also reviews the results of genomic approaches to characterize the regulon of each cell-specific s factor and the functions of some of the gene products. The best-characterized multicellular behaviors of B. subtilis, sporulation and the development of competence to take up exogenous DNA, are regulated by extracellular peptide signaling, analogous to M. xanthus A-signaling. Proteolysis is already known to play roles in A-, B-, and C-signaling during M. xanthus development, and it seems likely that many more roles will be uncovered, based on studies of B. subtilis. Just as these studies have provided a host of paradigms, so too will continued investigation of the myxobacteria and their neighbors continue to yield novel insights of medical, economic, and environmental benefit.

In stalked alphaproteobacteria such as Caulobacter crescentus, Asticcacaulis biprosthecum, and Hyphomicrobium neptunium, development is not triggered by environmental changes but is a consequence of normal progression through the cell cycle. In contrast to M. xanthus and B. subtilis, the oligotrophic C. crescentus has adopted a different strategy to cope with almost constant famine. First, the production of a chemotactically competent swarmer cell allows dispersal towards more nutritionally rich environments. Second, the successive synthesis of a flagellum, pili, and holdfast at the same pole of the cell optimizes attachment of swarmer cells to surfaces, thus improving the cell’s access to nutrients absorbed to surfaces. Finally, once attached tightly to a surface by its holdfast, the cell synthesizes a stalk, which dramatically improves nutrient uptake in the diffusion-limited environment where C. crescentus typically lives. This chapter focuses on the mechanisms of surface adhesion and stalk function, as well as how the overall developmental cycle is controlled. Differentiation is an essential process for adhesion of C. crescentus to surfaces. The stalk is an extension of the cell wall and membranes devoid of ribosomes, DNA, and cytoplasmic proteins. The stalk is transected perpendicularly by crossbands synthesized during each round of cell division, which may serve to compartmentalize the stalk from the cell body. A complex signal transduction pathway ensures that polar development, DNA replication, and cell division are coordinated. The new pole remains marked until late in cell division when TipN moves from the pole to the site of division.

Over 2 billion years ago, with the growth of large numbers of O2-producing cyanobacteria, our planet emerged from anaerobiosis. The cyanobacteria needed only light, water, and inorganic nutrients, with CO2 as a carbon source, to replicate. Numerous genes have been identified that are required specifically for synthesis and deposition of heterocyst envelope glycolipids and polysaccharide, respiratory processes in heterocysts, and cessation of division in those cells that will become heterocysts. Akinetes differentiate either specifically adjacent to heterocysts (or sometimes near but not contiguous), distant from heterocysts, or independent of heterocysts. Some nitrogen-fixing cyanobacteria establish symbiotic relationships with different plants and fungi. These cyanobacteria, mostly in the genus Nostoc, are able to form symbioses with bryophytes, ferns, gymnosperms, and angiosperms and provide fixed nitrogen to the plants. The expression of ntcA is dependent on the presence of an intact ntcA, and the expression of hetC may also be positively autoregulated. HetC is an unlikely candidate for a regulatory molecule, because its product is similar to ATP-binding cassette transporters of proteins, peptides, and polysaccharides. Cell wall metabolism evidently plays an important role in heterocyst formation, as was first shown by the observation that genes rfbP and rfbZ participate in the normal formation of vegetative cell lipopolysaccharide, and yet are Fox genes, required only for aerobic growth on N2.

The colony structure and life cycle of the grampositive, soil-dwelling bacterium Streptomyces coelicolor provide a fascinating exception to the view of bacteria as simple unicellular microorganisms. Mutations in genes involved in morphogenesis alter colony appearance but do not usually compromise viability. The majority of genes identified as being important for aerial hypha formation encode regulatory proteins; however, recent work has resulted in the characterization of two classes of structural molecules that are necessary for aerial development: the SapB surfactant peptide (specified by the ram gene cluster) and eight chaplin proteins (ChpA through H). It has been found that while Streptomyces has many of the conventional genes that are necessary for these processes to occur, Streptomyces cells are organized very differently from other bacteria and these differences are highly relevant to colony development and spore formation. In Streptomyces, however, DivIVA is an essential protein that does not seem to be associated with cell division but rather is crucial for coordinating cell wall growth. The basic mechanism of Z-ring formation appears to be shared between S. coelicolor and other prokaryotes; however, there are important differences in how Streptomyces employs and executes cell division. Streptomyces has no homologues of the Bacillus subtilis or Escherichia coli MinC or MinD proteins and uses its DivIVA for functions apparently unrelated to cell division.

The Dictyostelium cells that become stalk cells sacrifice themselves and die to help the spore cells get dispersed, and studies of how different strains either cooperate or cheat to become spore cells is helping to open this field. There are several human diseases or therapies for diseases that can be usefully studied using the simplicity of Dictyostelium to uncover molecular mechanisms. The cells accomplish this by secreting a glycoprotein called prestarvation factor (PSF). Insight into how such a quorum-sensing mechanism functions was gained when it was observed that D. discoideum cells starved at low cell densities differentiate only in buffer previously conditioned by a high density of starved cells. The cell-autonomous musical chairs cell-type choice mechanism causes prespore and prestalk cells to appear at random places within the mound. To maintain the proper ration of prestalk to prespore cells, prespore cells convert to anterior-like cells (ALC), which convert to PstO and then PstA cells. Differentiation-inducing factor (DIF)-1 induces the expression of prestalk genes and represses the expression of prespore genes. In addition to being an excellent organism to study chemotaxis, development, and differentiation, Dictyostelium is also used to understand the molecular basis of disease. One of the diseases under investigation is Legionnaires’ disease, which is caused by an infection by Legionella pneumophila bacteria. In humans, inhaled L. pneumophila is phagocytosed by macrophages present in the lungs. Dictyostelium uses both G-protein-coupled receptors and two-component receptors, while myxobacteria use two-component systems.

Most studies of biofilms have focused on single species and on genes that control or are regulated by life on a surface. As more information is uncovered by studies of pure cultures, these data can be applied towards understanding the roles of specific genes in multispecies interactions. This chapter focuses mostly on multi-species interactions among oral bacteria in biofilms: a few single-species biofilms are featured to discuss responses to environmental signals, including signals generated by the occupants within the biofilm. Signals involved in cell-to-cell communication among biofilm cells include acyl homoserine lactones, oligopeptides, and autoinducer-2 (AI-2). Importantly, an optimal concentration of 4,5-dihydroxy-2,3-pentanedione (DPD) was critical for maximal biofilm development. One site where natural multispecies biofilms are unusually accessible is the tooth surface in the human oral cavity. We use a retrievable enamel chip model system that permits us to place three pieces of enamel side by side in a groove cut into an acrylic stent that is placed bilaterally on the buccal surface of the lower dentition. The majority of cells in both sequentially and coaggregateinoculated biofilms were S. gordonii, regardless of the inoculation order. Usually biofilms are formed only on solid or semi-solid substrata, such as steel pipes or agar, respectively. Many environmental cues and signals likely govern multispecies biofilms that form and disperse. One of the most important determinants is the distance between signal generator and signal receiver.

This chapter presents a description of methods for cultivation of Myxococcus xanthus laboratory strains, as well as specific protocols for analysis of motility and development. Here the authors attempt to cover representative techniques, and to mention variations only occasionally. In nature, communities of M. xanthus reside in the soil on decaying plant material or herbivore dung and obtain nutrients by secreting digestive enzymes (such as proteases and lysozyme) to digest macromolecules from prey microorganisms or decaying organic matter. Initiation of the developmental program resulting in fruiting body formation requires (i) nutrient limitation, (ii) a solid surface, and (iii) sufficient population density. Fruiting body morphology and timing are greatly dependent on the medium surface. Therefore, solid media for development should be prepared the day before and plates should be cured (prewarmed) by incubating the plates with lids cracked open at least 20 min at 32ºC just before use. M. xanthus powers its movement over solid surfaces by using two genetically distinct motility systems. Social (S) motility—the coordinated movement of cells in large groups—predominates on soft and moist surfaces and is directly mediated by the extension and retraction of polar type IV pili. Adventurous (A) motility—the movement of single isolated cells—predominates on harder and drier surfaces and has been proposed to be based on either a jet engine-like extrusion of carbohydrate slime, a twisting or inching-like motion on the part of the M. xanthus cell, or intracellular motors pushing against dynamic focal adhesion points within the cell.

Analysis of gene expression is an important tool for the study of the program involved in development and sporulation of Myxococcus xanthus. A large number of genes have been fused to the β-galactosidase (lacZ) gene, and their expression profiles have been extensively studied using β-galactosidase assays. Recently, two new methods have become available, namely, quantitative PCR (QPCR) and microarray analysis. The QPCR method is feasible only for studying the expression of a limited number of genes (10 to 20 genes), whereas the strength of microarrays is their ability to monitor global gene expression. Microarrays are not suitable or cost-effective for studying gene expression of single genes, but they provide trends of global changes. For QPCR and microarray experiments the RNA must be of good quality; it should therefore be carefully checked for purity and integrity before use. The chapter focuses on the hybridization protocol for amino silane-coated arrays obtained from The Institute for Genomic Research (TIGR). The protocols should be used as starting points for these types of experiments, and some optimization will, especially for microarray experiments, be necessary.

Myxococcus xanthus has a remarkable repertoire of complex multicellular behaviors, including social gliding motility, predation, rippling, and fruiting body formation. There are a few genetic tools such as an autonomously replicating plasmid and a defined library of M. xanthus mutant strains that have yet to be developed to study these complex behaviors. This chapter contains a description of M. xanthus genetic tools and their practical applications. Generalized transducing particles form when by mistake a bacteriophage head or capsid assembles around a fragment of a donor bacterium’s chromosomal DNA or around a plasmid instead of the phage genome. When these particles infect a recipient host, the donor bacterium’s DNA is inserted into the recipient host and is free to undergo homologous recombination with the host cell chromosome. In M. xanthus, generalized transducing phages are typically used for genetic mapping and for strain constructions. The plasmids are introduced into M. xanthus cells, strains that carry the plasmid integrated into phage attachment sites in the chromosome are identified, and the appropriate assays are performed. Gene fusions have been used to assay the transcriptional/translational regulation of genes/proteins and to examine protein localization in bacterial cells. Fusions belong to one of two classes: transcriptional fusions or translational fusions. In the case of a transcriptional fusion, the reporter gene lacks a promoter, but it possesses a functional ribosome-binding site. In the case of a translational fusion, the reporter gene lacks a promoter and a functional ribosome-binding site.

This chapter summarizes research methods to encourage further research into the complex biology of Sorangium cellulosum. S. cellulosum is a cellulolytic myxobacterium that can grow on simple mineral medium with KNO3 as the sole nitrogen source and cellulose as the sole carbon source The authors have developed a reproducible protocol that induces fruiting body formation by S. cellulosum strain So ce56, which grows as dispersed cells in liquid. Distinct strains of S. cellulosum exhibit very different growth characteristics. The dispersed-growing So ce56 strain shows severely reduced swarming on several agar surfaces but does form fruiting bodies when sufficiently concentrated cell suspensions are used. They used the pMycoMarHyg transposon to construct developmental mutants of So ce56, and some of the resulting mutant phenotypes are shown in the chapter. Qualitative bioassays are useful tools for analysis of physiological differentiation defects exhibited by mutants. Chivosazole production of So ce56 was reported to depend on temperature, aeration, nitrogen source, and carbon source.