Despite the knowledge of complex prokaryotic-transcription mechanisms, generalized rules, such as the simplified organization of genes into operons with well-defined promoters and terminators, have had a significant role in systems analysis of regulatory logic in both bacteria and archaea. Here, we have investigated the prevalence of alternate regulatory mechanisms through genome-wide characterization of transcript structures of ∼64% of all genes, including putative non-coding RNAs in Halobacterium salinarum NRC-1. Our integrative analysis of transcriptome dynamics and protein–DNA interaction data sets showed widespread environment-dependent modulation of operon architectures, transcription initiation and termination inside coding sequences, and extensive overlap in 3′ ends of transcripts for many convergently transcribed genes. A significant fraction of these alternate transcriptional events correlate to binding locations of 11 transcription factors and regulators (TFs) inside operons and annotated genes—events usually considered spurious or non-functional. Using experimental validation, we illustrate the prevalence of overlapping genomic signals in archaeal transcription, casting doubt on the general perception of rigid boundaries between coding sequences and regulatory elements.

During evolution, enzyme-coding genes are acquired and/or replaced through lateral gene transfer and compiled into metabolic pathways. Gene regulatory networks evolve to fine tune biochemical fluxes through such metabolic pathways, enabling organisms to acclimate to nutrient fluctuations in a competitive environment. Here, we demonstrate that a single TrmB family transcription factor in Halobacterium salinarum NRC-1 globally coordinates functionally linked enzymes of diverse phylogeny in response to changes in carbon source availability. Specifically, during nutritional limitation, TrmB binds a cis-regulatory element to activate or repress 113 promoters of genes encoding enzymes in diverse metabolic pathways. By this mechanism, TrmB coordinates the expression of glycolysis, TCA cycle, and amino-acid biosynthesis pathways with the biosynthesis of their cognate cofactors (e.g. purine and thiamine). Notably, the TrmB-regulated metabolic network includes enzyme-coding genes that are uniquely archaeal as well as those that are conserved across all three domains of life. Simultaneous analysis of metabolic and gene regulatory network architectures suggests an ongoing process of co-evolution in which TrmB integrates the expression of metabolic enzyme-coding genes of diverse origins.

BackgroundRapidly characterizing the operational interrelationships among all genes in a given organism is a critical bottleneck to significantly advancing our understanding of thousands of newly sequenced microbial and eukaryotic species. While evolving technologies for global profiling of transcripts, proteins, and metabolites are making it possible to comprehensively survey cellular physiology in newly sequenced organisms, these experimental techniques have not kept pace with sequencing efforts. Compounding these technological challenges is the fact that individual experiments typically only stimulate relatively small-scale cellular responses, thus requiring numerous expensive experiments to survey the operational relationships among nearly all genetic elements. Therefore, a relatively quick and inexpensive strategy for observing changes in large fractions of the genetic elements is highly desirable.ResultsWe have discovered in the model organism Halobacterium salinarum NRC-1 that batch culturing in complex medium stimulates meaningful changes in the expression of approximately two thirds of all genes. While the majority of these changes occur during transition from rapid exponential growth to the stationary phase, several transient physiological states were detected beyond what has been previously observed. In sum, integrated analysis of transcript and metabolite changes has helped uncover growth phase-associated physiologies, operational interrelationships among two thirds of all genes, specialized functions for gene family members, waves of transcription factor activities, and growth phase associated cell morphology control.ConclusionsSimple laboratory culturing in complex medium can be enormously informative regarding the activities of and interrelationships among a large fraction of all genes in an organism. This also yields important baseline physiological context for designing specific perturbation experiments at different phases of growth. The integration of such growth and perturbation studies with measurements of associated environmental factor changes is a practical and economical route for the elucidation of comprehensive systems-level models of biological systems.

Xylella fastidiosa is a phytopathogenic bacterium that causes serious diseases in a wide range of economically important crops. Despite extensive comparative analyses of genome sequences of Xylella pathogenic strains from different plant hosts, nonpathogenic strains have not been studied. In this report, we show that X. fastidiosa strain J1a12, associated with citrus variegated chlorosis (CVC), is nonpathogenic when injected into citrus and tobacco plants. Furthermore, a DNA microarray-based comparison of J1a12 with 9a5c, a CVC strain that is highly pathogenic and had its genome completely sequenced, revealed that 14 coding sequences of strain 9a5c are absent or highly divergent in strain J1a12. Among them, we found an arginase and a fimbrial adhesin precursor of type III pilus, which were confirmed to be absent in the nonpathogenic strain by PCR and DNA sequencing. The absence of arginase can be correlated to the inability of J1a12 to multiply in host plants. This enzyme has been recently shown to act as a bacterial survival mechanism by down-regulating host nitric oxide production. The lack of the adhesin precursor gene is in accordance with the less aggregated phenotype observed for J1a12 cells growing in vitro. Thus, the absence of both genes can be associated with the failure of the J1a12 strain to establish and spread in citrus and tobacco plants. These results provide the first detailed comparison between a nonpathogenic strain and a pathogenic strain of X. fastidiosa, constituting an important step towards understanding the molecular basis of the disease.