The S. meliloti 1021 genome contains 14 genes for sigma factors [21], two of which code for RpoH sigma factors. However, rpoH1 and rpoH2 are not functionally

equivalent [22, 23]. The two genes are expressed differentially during growth in culture and during symbiosis, and only rpoH1 is required for growth in heat shock stress and for successful symbiosis with alfalfa [23, 24]. The presence of several copies of RpoH sigma factors suggests that rhizobia may find more respond more specifically to environmental changes and that the heat shock response could overlap the response to other stimuli [23]. Previous studies with S. meliloti revealed that an rpoH1 mutant exhibits increased sensitivity to various stress agents, including acid pH, suggesting that RpoH1 OICR-9429 manufacturer Temsirolimus cost is required to protect the bacterial cell against environmental stress encountered in solo or within the host [25]. Soil acidity constrains symbiotic nitrogen fixation and affects the exchange of molecular signals between rhizobia and their host, reducing nodulation [26–28]. Environmental pH stress constitutes therefore a limiting factor for S.

meliloti survival and development, both in the soil and in planta [29]. In a previous study, it was observed that the response to acidic pH stress in S. meliloti is versatile and characterized by the differential expression of whole sets of genes associated with various cellular functions, such as exopolysaccharide I biosynthesis and chemotaxis Cytidine deaminase [30]. The purpose of the present study was to gain detailed insight

into the complex stress response regulatory system of S. meliloti using pH stress as an effector and to verify if specific sigma factors in S. meliloti are involved in pH stress response. Our aim was likewise to provide a basis for understanding the molecular mechanisms of sigma factor regulation and identify genes involved in pH stress response whose expression is sigma factor-dependent. Because the regulation of gene expression is a dynamic process, special attention was granted to the characterization of changes in gene expression over time. Results Identification of sigma factors involved in the pH stress response of S. meliloti To explore the role of sigma factors in S. meliloti under acidic pH stress conditions, marker-free deletion mutants were successfully produced for the sigma factor genes rpoE1, rpoE2, rpoE5, rpoH1 and fecI, with the utilization of gene Splicing by Overlap Extension or gene SOEing technique [31]. Those sigma factor genes were chosen for mutant constructions for, based on amino acid sequence comparison analysis, they represent the three main functional classes of alternative sigma factors, namely extracytoplasmic function, heat shock and iron metabolism control.