Also, it enabled us to extract the fundamental patterns of gene expression inherent in the data. In S. meliloti, two RpoH-type sigma factors are annotated in the genome . RpoH1 and RpoH2 are involved in different stress responses, and this probably provides increased capacity for S. meliloti to adapt to different environments. We suggest for the first time that RpoH1 efficiently regulates the expression of specific heat shock genes in response
to pH stress in S. meliloti. This type of regulation structure would also be efficient for adjustment to other stresses requiring rapid change of metabolic mode as well as thermal adaptation. We ultimately conclude that RpoH1 is necessary for the dynamic response of S. meliloti to sudden Staurosporine mouse pH shift and it accounts for critical changes in gene expression during pH stress response. These findings form a basis for subsequent analyses
of regulation and function of the stress response in S. meliloti. The time-course study provides efficient methodology for hypothesis-driven investigations to dissect the roles of sigma factors and other key players in transcription regulation not only in pH stress conditions, but in general stress response and adaptation. In addition to the recognition of individual genes with altered expressions, the proposed method for clustering of time-course check details data enabled us to identify gene clusters, each with a unique time-dependent expression pattern. Further
biochemical and genetic studies not on the regulatory events of S. meliloti cells undergoing environmental stress should continue to provide useful information for further understanding of the role of RpoH1 and other alternative sigma factors in stress response. Conclusions Our study indicated that sigma factor RpoH1 plays an important role in the response to low pH stress in S. meliloti. This role was efficiently unraveled by time-course microarray studies, in which key players involved in stress response whose transcription is under regulation of RpoH1 were identified. Clustering of time-course microarray data of S. meliloti wild type and rpoH1 mutant allowed for the classification of three groups of genes that were transcriptionally regulated upon pH stress in an RpoH1-independent, in an RpoH1-dependent or in a complex manner. Among the genes that showed an RpoH1-dependent regulation, there were several coding for heat shock and chaperone proteins. Time-course global gene expression analyses can be further employed to facilitate the temporal study of regulatory mechanisms and provide a more comprehensive framework for studying dynamic cellular processes, such as stress response. Methods Bacterial strains, plasmids, and growth conditions The bacterial strains and plasmids used in this work are listed in Table 1. E.