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Myxococcus xanthus copes with starvation by producing fruiting bodies filled with dormant and stress-resistant spores. Here, we aimed to better define the gene regulatory network associated with Nla28, a transcriptional activator/enhancer binding protein (EBP) and a key regulator of the early starvation response. Previous work showed that Nla28 directly regulates EBP genes that are important for fruiting body development. However, the Nla28 regulatory network is likely to be much larger because hundreds of starvation-induced genes are downregulated in a nla28 mutant strain. To identify candidates for direct Nla28-mediated transcription, we analyzed the downregulated genes using a bioinformatics approach. Nine potential Nla28 target promoters (29 genes) were discovered. The results of in vitro promoter binding assays, coupled with in vitro and in vivo mutational analyses, suggested that the nine promoters along with three previously identified EBP gene promoters were indeed in vivo targets of Nla28. These results also suggested that Nla28 used tandem, imperfect repeats of an 8-bp sequence for promoter binding. Interestingly, eight of the new Nla28 target promoters were predicted to be intragenic. Based on mutational analyses, the newly identified Nla28 target loci contained at least one gene that was important for starvation-induced development. Most of these loci contained genes predicted to be involved in metabolic or defense-related functions. Using the consensus Nla28 binding sequence, bioinformatics, and expression profiling, 58 additional promoters and 102 genes were tagged as potential Nla28 targets. Among these putative Nla28 targets, functions, such as regulatory, metabolic, and cell envelope biogenesis, were assigned to many genes. IMPORTANCE In bacteria, starvation leads to profound changes in behavior and physiology. Some of these changes have economic and health implications because the starvation response has been linked to the formation of biofilms, virulence, and antibiotic resistance. To better understand how starvation contributes to changes in bacterial physiology and resistance, we identified the putative starvation-induced gene regulatory network associated with Nla28, a transcriptional activator from the bacterium Myxoccocus xanthus. We determined the mechanism by which starvation-responsive genes were activated by Nla28 and showed that several of the genes were important for the formation of a highly resistant cell type.

Myxococcus xanthus is a bacterium that lives on surfaces as a predatory biofilm called a swarm. As a growing swarm feeds on prey and expands, it displays dynamic multicellular patterns such as traveling waves called ripples and branching protrusions called flares. The rate at which a swarm expands across a surface, and the emergence of the coexisting patterns, are all controlled through coordinated cell movement. M. xanthus cells move using two motility systems known as adventurous (A) and social (S). Both are involved in swarm expansion and pattern formation. In this study, we describe a set of M. xanthus swarming genotype-to-phenotype associations that include both genetic and environmental perturbations. We identified new features of the swarming phenotype, recorded and measured swarm expansion using time-lapse microscopy, and compared the impact of mutations on different surfaces. These observations and analyses have increased our ability to discriminate between swarming phenotypes and provided context that allows us to identify some phenotypes as improbable outliers within the M. xanthus swarming phenome. IMPORTANCE Myxococcus xanthus grows on surfaces as a predatory biofilm called a swarm. In nature, a feeding swarm expands by moving over and consuming prey bacteria. In the laboratory, a swarm is created by spotting cell suspension onto nutrient agar in lieu of prey. The suspended cells quickly settle on the surface as the liquid is absorbed into the agar, and the new swarm then expands radially. An assay that measures the expansion rate of a swarm of mutant cells is the first, and sometimes only, measurement used to decide whether a particular mutation impacts swarm motility. We have broadened the scope of this assay by increasing the accuracy of measurements and introducing prey, resulting in new identifiable and quantifiable features that can be used to improve genotype-to-phenotype associations.

The Clp/Hsp100 proteins are chaperones that play a role in protein degradation and reactivation. In bacteria, they exhibit a high degree of pleiotropy, affecting both individual and multicellular phenotypes. In this article, we present the first characterization of a Clp/Hsp100 homolog in Myxococcus xanthus (MXAN_4832 gene locus). Deletion of MXAN_4832 causes defects in both swarming and aggregation related to cell motility and the production of fibrils, which are an important component of the extracellular matrix of a swarm. The deletion also affects the formation of myxospores during development, causing them to become sensitive to heat. The protein product of MXAN_4832 can act as a chaperone in vitro, providing biochemical evidence in support of our hypothesis that MXAN_4832 is a functional Clp/Hsp100 homolog. There are a total of 12 Clp/Hsp100 homologs in M. xanthus, including MXAN_4832, and, based on its mutational and biochemical characterization, they may well represent an important group.

Under starvation conditions, a swarm of Myxococcus xanthus cells will undergo development, a multicellular process culminating in the formation of many aggregates called fruiting bodies, each of which contains up to 100,000 spores. The mechanics of symmetry breaking and the self-organization of cells into fruiting bodies is an active area of research. Here we use microcinematography and automated image processing to quantify several transient features of developmental dynamics. An analysis of experimental data indicates that aggregation reaches its steady state in a highly nonmonotonic fashion. The number of aggregates rapidly peaks at a value 2- to 3-fold higher than the final value and then decreases before reaching a steady state. The time dependence of aggregate size is also nonmonotonic, but to a lesser extent: average aggregate size increases from the onset of aggregation to between 10 and 15 h and then gradually decreases thereafter. During this process, the distribution of aggregates transitions from a nearly random state early in development to a more ordered state later in development. A comparison of experimental results to a mathematical model based on the traffic jam hypothesis indicates that the model fails to reproduce these dynamic features of aggregation, even though it accurately describes its final outcome. The dynamic features of M. xanthus aggregation uncovered in this study impose severe constraints on its underlying mechanisms.

We have characterized and quantified a form of bacterial chemotaxis that manifests only as an emergent property by measuring symmetry breaking in a swarm of Myxococcus xanthus exposed to a two-dimensional nutrient gradient from within an agar substrate. M. xanthus chemotaxis requires cell-cell contact and coordinated motility, as individual motile cells exhibit only nonvectorial movement in the presence of a nutrient gradient. Genes that specifically affect M. xanthus chemotaxis include at least 10 of the 53 that express enhancer binding proteins of the NtrC-like class, an indication that this behavior is controlled through transcription, most likely by a complex signal transduction network.

Microcinematography was used to examine fruiting body development of Myxococcus xanthus. Wild-type cells progress through three distinct phases: a quiescent phase with some motility but little aggregation (0 to 8 h), a period of vigorous motility leading to raised fruiting bodies (8 to 16 h), and a period of maturation during which sporulation is initiated (16 to 48 h). Fruiting bodies are extended vertically in a series of tiers, each involving the addition of a cell monolayer on top of the uppermost layer. A pilA (MXAN_5783) mutant produced less extracellular matrix material and thus allowed closer examination of tiered aggregate formation. A csgA (MXAN_1294) mutant exhibited no quiescent phase, aberrant aggregation in phase 2, and disintegration of the fruiting bodies in the third phase.

A search of the M1genome sequence, which includes 97% of the Myxococcus xanthus genes, identified 53 sequence homologs of sigma54-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.

The multicellular developmental cycle of Myxococcus xanthus requires large-scale changes in gene transcription, and recent findings indicate that NtrC-like activators play a prominent role in regulating these changes. In this study, we made insertions in 28 uncharacterized ntrC-like activator (nla) genes and found that eight of these insertions cause developmental defects. Hence, these results are consistent with the idea that M. xanthus uses a series of different NtrC-like activators during fruiting body development. Four of the eight developmental mutants we identified have motility defects. The nla1, nla19, and nla23 mutants show S-motility defects, while the nla24 mutant shows defects in both S-motility and A-motility. During development, aggregation of the nla1, nla19, and nla23 mutants is delayed slightly and the nla24 mutant shows no signs of aggregation or sporulation. The nla4, nla6, nla18, and nla28 mutants have no appreciable loss in motility, but they fail to aggregate and to sporulate normally. The nla18 mutant belongs to a special class of developmental mutants whose defects can be rescued when they are codeveloped with wild-type cells, suggesting that nla18 fails to produce a cell-cell signal required for development. The three remaining activator mutants, nla4, nla6, and nla28, appear to have complex developmental phenotypes that include deficiencies in cell-cell developmental signals.