Development and application of differential display reverse: Transcriptase-polymerase chain reaction to examine conditional gene expression in Ruminococcus albus

The studies presented herein represent some of the initial steps towards developing a mechanistic understanding of the physiological response of Ruminococcus albus to phenylacetic acid (PAA) and phenylpropionic acid (PPA). These phenyl-substituted fatty acids are required for effective cellulose hydrolysis by R. albus, one of the three predominant cellulolytic ruminal bacteria. The gene encoding a novel form of a cellulose-binding protein (CbpC) was isolated from Ruminococcus albus 8. CbpC possesses an amino-terminal motif characteristic of the Pil family of proteins, which are most commonly involved with the formation of type 4 fimbriae, and the remainder of this protein sequence was found to have significant identity with motifs in the surface antigen protein of Rickettsia spp. which are involved in host cell recognition and invasion. Downstream of the cbpC gene are several open reading frames that possess notable amino acid identity to type II secretion system components. CbpC mRNA and protein abundance is greater in cellulose-grown cells relative to cellobiose-grown cells. In addition, PAA/PPA or ruminal fluid increase cbpC mRNA abundance in cellobiose-grown cells. One of the differentially expressed sequence tags obtained in differential display reverse transcriptase-polymerase chain reaction (DD RT-PCR), in response to ruminal fluid or PAA/PPA, revealed a four-cistron operon, parABCD (phenyl-substituted acid responsive) which encodes a protein with notable sequence similarity to members of the LacI-GalR family regulatory proteins, in addition to the structural components of a binding protein-dependent uptake system. The four transcripts derived from two promoters in the parABCD operon are all expressed in greater abundance, with the inclusion of ruminal fluid or PAA/PPA. DNA mobility shift assays performed with R. albus cell extracts suggested the presence of cis-acting elements affecting transcription from the P1 promoter upstream of the ParA open reading frame