Global gene expression analysis of the Myxococcus xanthus developmental time course.

To accurately identify the genes and pathways involved in the initiation of the Myxococcus xanthus multicellular developmental program, we have previously reported a method of growing vegetative populations as biofilms within a controllable environment. Using a modified approach to remove up to ~90% rRNAs, we report a comprehensive transcriptional analysis of the M. xanthus developmental cycle while comparing it with the vegetative biofilms grown in rich and poor nutrients. This study identified 1522 differentially regulated genes distributed within eight clusters during development. It also provided a comprehensive overview of genes expressed during a nutrient-stress response, specific development time points, and during development initiation and regulation. We identified several differentially expressed genes involved in key central metabolic pathways suggesting their role in regulating myxobacterial development. Overall, this study will prove an important resource for myxobacterial researchers to delineate the regulatory and functional pathways responsible for development from those of the general nutrient stress response.

Peripheral rods: a specialized developmental cell type in Myxococcus xanthus.

In response to nutrient deprivation, the ubiquitous Gram-negative soil bacterium Myxococcus xanthus undergoes a well-characterized developmental response, resulting in the formation of a multicellular fruiting body. The center of the fruiting body consists of myxospores; surrounding this structure are rod-shaped peripheral cells. Unlike spores, the peripheral rods are a metabolically active cell type that inhabits nutrient-deprived environments. The survival characteristics exhibited by peripheral rods, protection from oxidative stress and heat shock, are common survival characteristics exhibited by cells in stationary phase including modifications to morphology and metabolism. Vegetative M. xanthus cells undergo a number of physiological changes during the transition into stationary phase similar to other proteobacteria. In M. xanthus, stationary-phase cells are not considered a component of the developmental response and occur when cells are grown on nutrient-rich plates or in dispersed aqueous media. However, this cell type is not routinely studied and little of its physiology is known. Similarities between these two stress-induced cell types led to the question of whether peripheral rods are actually a distinct developmental cell type or simply cells in stationary phase. In this study, we examine the transcriptome of peripheral rods and its relationship to development. This work demonstrates that peripheral rods are in fact a distinct developmentally differentiated cell type. Although peripheral rods and stationary phase cells display similar characteristics, each transcriptomic pattern is unique and quite different from that of any other M. xanthus cell type.

A Minimal Threshold of c-di-GMP Is Essential for Fruiting Body Formation and Sporulation in Myxococcus xanthus.

Generally, the second messenger bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) regulates the switch between motile and sessile lifestyles in bacteria. Here, we show that c-di-GMP is an essential regulator of multicellular development in the social bacterium Myxococcus xanthus. In response to starvation, M. xanthus initiates a developmental program that culminates in formation of spore-filled fruiting bodies. We show that c-di-GMP accumulates at elevated levels during development and that this increase is essential for completion of development whereas excess c-di-GMP does not interfere with development. MXAN3735 (renamed DmxB) is identified as a diguanylate cyclase that only functions during development and is responsible for this increased c-di-GMP accumulation. DmxB synthesis is induced in response to starvation, thereby restricting DmxB activity to development. DmxB is essential for development and functions downstream of the Dif chemosensory system to stimulate exopolysaccharide accumulation by inducing transcription of a subset of the genes encoding proteins involved in exopolysaccharide synthesis. The developmental defects in the dmxB mutant are non-cell autonomous and rescued by co-development with a strain proficient in exopolysaccharide synthesis, suggesting reduced exopolysaccharide accumulation as the causative defect in this mutant. The NtrC-like transcriptional regulator EpsI/Nla24, which is required for exopolysaccharide accumulation, is identified as a c-di-GMP receptor, and thus a putative target for DmxB generated c-di-GMP. Because DmxB can be-at least partially-functionally replaced by a heterologous diguanylate cyclase, these results altogether suggest a model in which a minimum threshold level of c-di-GMP is essential for the successful completion of multicellular development in M. xanthus.

Growth of Myxococcus xanthus in continuous-flow-cell bioreactors as a method for studying development.

Nutrient sensors and developmental timers are two classes of genes vital to the establishment of early development in the social soil bacterium Myxococcus xanthus. The products of these genes trigger and regulate the earliest events that drive the colony from a vegetative state to aggregates, which ultimately leads to the formation of fruiting bodies and the cellular differentiation of the individual cells. In order to more accurately identify the genes and pathways involved in the initiation of this multicellular developmental program in M. xanthus, we adapted a method of growing vegetative populations within a constant controllable environment by using flow cell bioreactors, or flow cells. By establishing an M. xanthus community within a flow cell, we are able to test developmental responses to changes in the environment with fewer concerns for effects due to nutrient depletion or bacterial waste production. This approach allows for greater sensitivity in investigating communal environmental responses, such as nutrient sensing. To demonstrate the versatility of our growth environment, we carried out time-lapse confocal laser scanning microscopy to visualize M. xanthus biofilm growth and fruiting body development, as well as fluorescence staining of exopolysaccharides deposited by biofilms. We also employed the flow cells in a nutrient titration to determine the minimum concentration required to sustain vegetative growth. Our data show that by using a flow cell, M. xanthus can be held in a vegetative growth state at low nutrient concentrations for long periods, and then, by slightly decreasing the nutrient concentration, cells can be allowed to initiate the developmental program.

The FsrA sRNA and FbpB protein mediate the iron-dependent induction of the Bacillus subtilis lutABC iron-sulfur-containing oxidases.

The Bacillus subtilis ferric uptake regulator (Fur) protein regulates iron homeostasis and directly represses more than 20 operons. Fur indirectly regulates many more genes, including those controlled by the small, noncoding RNA FsrA. FsrA translationally represses numerous target genes and, for at least some targets, appears to function in conjunction with one or more of three small, basic proteins, known as FbpA, FbpB, and FbpC. The lactate-inducible lutABC operon encodes iron sulfur-containing enzymes required for growth on lactate. We here demonstrate that a fur mutant strain grows poorly on lactate due to FsrA-dependent repression of LutABC synthesis. Growth is restored in an fsrA mutant and also partially restored by mutation of the fbpAB operon. Genetic studies indicate that the 48-amino-acid FbpB protein but not FbpA contributes to regulation of lutABC. FbpB may function, at least in part, by increasing the efficiency of FsrA targeting to the lutABC mRNA, since the role of FbpB can be bypassed by modest upregulation of FsrA. These results provide support for a model in which FbpB, and perhaps other Fbp proteins, contributes along with FsrA to the translational regulation of gene expression.

A global investigation of the Bacillus subtilis iron-sparing response identifies major changes in metabolism.

The Bacillus subtilis ferric uptake regulator (Fur) protein is the major sensor of cellular iron status. When iron is limiting for growth, derepression of the Fur regulon increases the cellular capacity for iron uptake and mobilizes an iron-sparing response mediated in large part by a small noncoding RNA named FsrA. FsrA functions, in collaboration with three small basic proteins (FbpABC), to repress many "low-priority" iron-containing enzymes. We have used transcriptome analyses to gain insights into the scope of the iron-sparing response and to define subsets of genes dependent for their repression on FsrA, FbpAB, and/or FbpC. Enzymes of the tricarboxylic acid (TCA) cycle, including aconitase and succinate dehydrogenase (SDH), are major targets of FsrA-mediated repression, and as a consequence, flux through this pathway is significantly decreased in a fur mutant. FsrA also represses the DctP dicarboxylate permease and the iron-sulfur-containing enzyme glutamate synthase (GltAB), which serves as a central link between carbon and nitrogen metabolism. Allele-specific suppression analysis was used to document a direct RNA-RNA interaction between the FsrA small RNA (sRNA) and the gltAB leader region. We further demonstrated that distinct regions of FsrA are required for the translational repression of the GltAB and SDH enzyme complexes.

The Bacillus subtilis iron-sparing response is mediated by a Fur-regulated small RNA and three small, basic proteins.

Regulation of bacterial iron homeostasis is often controlled by the iron-sensing ferric uptake repressor (Fur). The Bacillus subtilis Fur protein acts as an iron-dependent repressor for siderophore biosynthesis and iron transport proteins. Here, we demonstrate that Fur also coordinates an iron-sparing response that acts to repress the expression of iron-rich proteins when iron is limiting. When Fur is inactive, numerous iron-containing proteins are down-regulated, including succinate dehydrogenase, aconitase, cytochromes, and biosynthetic enzymes for heme, cysteine, and branched chain amino acids. As a result, a fur mutant grows slowly in a variety of nutrient conditions. Depending on the growth medium, rapid growth can be restored by mutations in one or more of the molecular effectors of the iron-sparing response. These effectors include the products of three Fur-regulated operons that encode a small RNA (FsrA) and three small, basic proteins (FbpA, FbpB, and FbpC). Extensive complementarity between FsrA and the leader region of the succinate dehydrogenase operon is consistent with an RNA-mediated translational repression mechanism for this target. Thus, iron deprivation in B. subtilis activates pathways to remodel the proteome to preserve iron for the most critical cellular functions.

CsoR regulates the copper efflux operon copZA in Bacillus subtilis.

The adaptation of Bacillus subtilis to elevated levels of copper ions requires the copper-inducible copZA operon encoding a copper chaperone and efflux ATPase. Here we identify CsoR (formerly YvgZ) as the copper-sensing repressor that regulates the copZA operon. CsoR binds with high affinity to an operator site overlapping the copZA promoter and its binding is specifically inhibited by copper salts. As previously described, the YhdQ (CueR) protein also binds to the copZA regulatory region, but genetic experiments indicate that this protein is not responsible for the copper-dependent regulation of this operon.