Identification and Characterization of RpoS Regulon and RpoS-Dependent Promoters in Burkholderia pseudomallei

Abstract

RpoS subunit of RNA polymerase is a bacterial alternative sigma factor and major regulator important for response to a variety of stress conditions. However, RpoS-dependent genes in Burkholderia pseudomallei remained undefined. We identified the RpoS regulon of B. pseudomallei using a proteomics approach, which revealed 70 differentially expressed proteins between rpoS(+) and rpoS(-) strains. The RpoS-dependent genes were then classified into 14 functional categories, most of which were related to stress response. We then used Hidden Markov Model (HMM) for prediction of RpoS-dependent promoters in 51 genes encoding 63 down-regulated proteins in rpoS(-) strain and successfully defined such promoters, which were classified into three main groups based upon their consensus sequences. Groups 1 and 2, which had the highest potential, were mostly "stress response" genes, whereas Group 3, with the lowest potential, belonged to genes encoding "hypothetical proteins". The promoter prediction was confirmed by 5'-RACE-PCR sequencing. We also expanded and defined RpoS-controlled genes from RpoS regulon, based on proteomic data, RpoS-dependent promoter prediction, gene organization and operon prediction. We finally confirmed the co-expression of these RpoS-dependent genes in operons using RT-PCR. Our data lead to better understanding of RpoS regulation in B. pseudomallei, whose RpoS regulon differs from other Gram-negative bacteria.

A Proteome Reference Map of the Causative Agent of Melioidosis Burkholderia pseudomallei

“…Type III secretion-related proteins are required for invasion of pathogenic bacteria into host cells. Expression of many of these proteins has been shown to be controlled by stress response sigma factors RpoS and RpoE in B. pseudomallei [8, 9]. Thus, translating our proteome reference map to other comparative proteome studies of B. pseudomallei grown at early stationary phase shall benefit the study of pathogenesis caused by B. pseudomallei , since expression of these pathogenesis-related proteins might be regulated by bacterial intrinsic factors and growth condition.…”

“…The clinical isolated B. pseudomallei strain 844 [12] was grown in 100 mL Luria Bertani (LB) medium at 37°C and 200 rpm [9]. Bacterial cells were pelleted using the culture at the 12th h of growth (OD 600 of about 9.0) corresponding to the early stationary phase by centrifugation at 10,000 ×g and 4°C.…”

“…2DE was performed as previously described [9]. 2DE gels were fixed in fixing solution (50% v/v ethanol and 2% v/v phosphoric acid) for 1 h. The gels were washed by soaking in distilled water for 10 min.…”

Strategies for Intracellular Survival of Burkholderia pseudomallei

“…However, the rpoS mutant was not tested for its ability to move intracellularly by actin-based motility (Table 1). The RpoS regulon in B. pseudomallei has been elucidated by comparing protein expression in wild-type and rpoS mutant strains; 70 proteins (derived from 58 unique genes) showed differential expression of >3-fold ( p  < 0.05) between the two strains (Osiriphun et al, 2009). The proteins encoded by these differentially expressed genes included bacterial stress response and cell envelope biogenesis components as well as some putative virulence factors, including a T3SS-1 component.…”

The RpoS Sigma Factor Negatively Regulates Production of IAA and Siderophore in a Biocontrol Rhizobacterium, Pseudomonas chlororaphis O6

“…In contrast, two proteins involved in the cellular utilization of iron from heme sources, heme oxygenase (HemeO) and a TonB-dependent membrane heme receptor (CirA) were detected at higher levels in the mutant (Table 1). RpoS regulated genes and proteins were identified in Geobacter sulfurreducens and Burkholderia pseudomallei (Nunez et al, 2006; Osiriphun et al, 2009). As expected, most of the RpoS regulated genes and proteins are related to stress responses, but many of the RpoS regulated genes and proteins are involved in the expression of diverse physiological traits, such as general metabolism, transcription, translation, cell motility and secretions (Nunez et al, 2006; Osiriphun et al, 2009).…”