Differential protein phosphorylation-dephosphorylation in response to carbon source in Ruminococcus flavefaciens FD-1.

AIMS: The aims of this study were to study the effect of cellobiose or cellulose as a carbon source on the differential protein phosphorylation-dephosphorylation of cytoplasmic and membrane-associated proteins from Ruminococcus flavefaciens FD-1. METHODS AND RESULTS: SDS-PAGE analysis was used to compare in vitro labelled proteins (32P-ATP) isolated from R. flavefaciens FD-1 grown on either cellobiose or cellulose as the carbon source. Distinctly different protein phosphorylation patterns were detected depending on carbon source and cell fraction. Analysis of the nature of the phosphorylated proteins indicates that phosphorylated proteins from cellobiose grown cultures are phosphorylated on serine residues, whereas phosphorylated proteins from cellulose grown cultures are phosphorylated on threonine residues. CONCLUSIONS: The results of this comparative analysis show a shift from serine phosphorylation of proteins to a threonine phosphorylation when R. flavefaciens FD-1 cells are grown on cellulose as opposed to cellobiose. There appears to be a role for these phosphorylation events in sensing the carbon source for growth and regulating co-ordinated metabolism in R. flavefaciens FD-1. SIGNIFICANCE AND IMPACT OF THE STUDY: We have demonstrated that there is a protein phosphorylation system in R. flavefaciens FD-1 that may be the primary sensing system for carbon source by R. flavefaciens FD-1 and the further regulation of gene expression related to cellulose degradation.

Identification of patterns of transmission of Salmonella within swine production systems using pulsed field gel electrophoresis (PFGE) and repetitive sequence polymerase chain reaction (REP-PCR): a quantitative analysis.

Pulsed field gel electrophoresis (PFGE) using 3 enzymes (Spe I, Xba I, Avr II) and repetitive sequence polymerase chain reaction (REP-PCR) with 3 primers (BOX, ERIC, REP) were compared with respect to their validity as a method for identifying transmission of Salmonella on swine farms. Sixty-eight isolates of Salmonella were obtained from feces of swine, cats, mice, and birds, insect body parts, water and floor samples, and boot scrapings collected on 9 swine farms in Illinois USA. Genetic distances between isolates were calculated using the Dice matching coefficient. Cluster analysis of distance matrices was conducted using the UPG-MA algorithm. There was no significant difference between PFGE and REP-PCR in the genetic diversity detected; however, REP-PCR differentiated between 14 pairs of isolates which PFGE identified as identical. There were no significant differences between PFGE and REP-PCR in identifying all or most close genetic links as isolates from the same farm, the same building, and from the same sampling visit, suggesting ecological validity for both methods. Thus, REP-PCR should be considered as an acceptable and perhaps preferable alternative to PFGE as a genotyping method for studies of Salmonella transmission.

Molecular cloning, sequencing, and expression of a novel multidomain mannanase gene from Thermoanaerobacterium polysaccharolyticum.

The manA gene of Thermoanaerobacterium polysaccharolyticum was cloned in Escherichia coli. The open reading frame of manA is composed of 3,291 bases and codes for a preprotein of 1,097 amino acids with an estimated molecular mass of 119,627 Da. The start codon is preceded by a strong putative ribosome binding site (TAAGGCGGTG) and a putative -35 (TTCGC) and -10 (TAAAAT) promoter sequence. The ManA of T. polysaccharolyticum is a modular protein. Sequence comparison and biochemical analyses demonstrate the presence of an N-terminal leader peptide, and three other domains in the following order: a putative mannanase-cellulase catalytic domain, cellulose binding domains 1 (CBD1) and CBD2, and a surface-layer-like protein region (SLH-1, SLH-2, and SLH-3). The CBD domains show no sequence homology to any cellulose binding domain yet reported, hence suggesting a novel CBD. The duplicated CBDs, which lack a disulfide bridge, exhibit 69% identity, and their deletion resulted in both failure to bind to cellulose and an apparent loss of carboxymethyl cellulase and mannanase activities. At the C-terminal region of the gene are three repeats of 59, 67, and 56 amino acids which are homologous to conserved sequences found in the S-layer-associated regions within the xylanases and cellulases of thermophilic members of the Bacillus-Clostridium cluster. The ManA of T. polysaccharolyticum, besides being an extremely active enzyme, is the only mannanase gene cloned which shows this domain structure.

Complete nucleotide sequence of a cryptic plasmid, pBAW301, from the ruminal anaerobe Ruminococcus flavefaciens R13e2.

The complete nucleotide sequence of a cryptic plasmid designated pBAW301, from the Gram-positive ruminal bacterium Ruminococcus flavefaciens R13e2, has been determined. This plasmid is 1768 bp in size and has an overall G+C content of 43.5%. Computer analysis of the sequence data revealed an open reading frame, ORF1 (256 amino acids), which is similar to the Rep protein of the Bacillus borstelensis plasmid pHT926. ORF1 is preceded by Shine-Dalgarno and Escherichia coli-10 and -35 like sequences. Nine smaller open reading frames showed no significant homologies to known protein sequences. Analysis of replication intermediates and the nucleotide sequence indicate that the plasmid does not replicate by a rolling-circle mode of replication similar to other plasmids from Gram-positive bacteria. Moreover, sequences typical of theta replication origins were not found in the nucleotide sequence of pBAW301. These data suggest that this plasmid either replicates by an as yet undescribed mechanism, or represents a new class of theta replicating plasmids.

Cold-sensitive mutations in beta and beta' subunit gene affecting the interaction of RNA polymerase with promoters.

RNA polymerases with a cold-sensitive activity were purified from seven mutants of Escherichia coli. Subunit reconstitution experiments have shown that RNA polymerases from three mutants (Rpob262, RpoB264, and RpoB265) owed their cold sensitivity to alterations in the beta subunit. Three mutants (RpoC3, RpoC263, and RpoC267) were shown to be defective in the beta' subunit and one (RpoBC266) in both beta and beta' subunits. Two mutations (rpoC3 and rpoC263) reduced the level of RNA polymerase reconstitution. RNA polymerases from RpoC3 and RpoBC266 mutants are defective in RNA chain elongation at 6 degree C, while all the other mutants are defective in RNA polymerase-promoter interaction. most mutant RNA polymerases differ from the wild-type enzyme in transcription selectivity. The results obtained in this study indicate that both beta and beta' subunits are involved in RNA chain elongation and promoter binding and selection.