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ABSTRACT 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. 

ABSTRACT 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.