Albusin B, a Bacteriocin from the Ruminal Bacterium Ruminococcus albus 7 That Inhibits Growth of Ruminococcus flavefaciens

Abstract

An ϳ32-kDa protein (albusin B) that inhibited growth of Ruminococcus flavefaciens FD-1 was isolated from culture supernatants of Ruminococcus albus 7. Traditional cloning and gene-walking PCR techniques revealed an open reading frame (albB) encoding a protein with a predicted molecular mass of 32,168 Da. A BLAST search revealed two homologs of AlbB from the unfinished genome of R. albus 8 and moderate similarity to LlpA, a recently described 30-kDa bacteriocin from Pseudomonas sp. strain BW11M1.Ruminococcus albus and Ruminococcus flavefaciens are important cellulose-degrading bacteria in the rumen (6). R. albus has been reported to outnumber R. flavefaciens in defined laboratory cocultures (4,16,17,22) and in the rumens of sheep (25) and dairy cattle (28). Several reports suggested that predominance of R. albus may be due to its production of bacteriocin-like substances (4,16,17), but none of these agents had been purified. The objective of this study was to purify and characterize bacteriocin-like substances produced by R. albus.Culture supernatants of R. albus 7 grown in modified Dehority medium (27) with cellulose or cellobiose as a fermentable carbohydrate inhibited growth of R. flavefaciens FD-1 when assayed by a plate culture assay (4). Because most of the bacteriocins purified from gram-positive bacteria have low molecular masses (Ͻ10 kDa), we examined filtrates of R. albus 7 cultures for inhibitory activity toward R. flavefaciens. Cultures grown in 10 liters of modified Dehority medium with cellobiose were passed through a 0.2-m-pore-size hollow fiber cartridge, and the filtrate was passed through a 10-kDa-molecular-masscutoff cartridge. Substantial inhibitory activity was observed in the nominal 10-kDa filtrate, but surprisingly this filtrate contained a 32-kDa protein (referred to here as albusin B) that was ultimately shown to account for most of the activity. Although the retentate from the 10-kDa cartridge also contained an inhibitory protein of 32 kDa, the mixture of components in the retentate was far more complex than that in the filtrate. The simple protein profile of the filtrate facilitated purification of albusin B, which was accomplished by a process that also included ammonium sulfate precipitation (40 to 60% saturation range) and a BioGel P6 gel filtration column (Table 1). Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis revealed a single protein band (32 kDa) from the P6 column (Fig. 1). While the purification was facilitated by the 10-kDa-molecular-mass-cutoff ultrafiltration step, the complete protocol resulted in only a 4.7% activity yield and an estimated purification of 220-fold. Because some of the inhibitory activity in culture supernatants was due to a second inhibitor (albusin A, which will be described elsewhere), whose concentration declined during the course of the purification, the extent of purification of albusin B must be regarded as a lower bound.All seven R. flavefaciens strains tested (FD-1, B34b, 17, C94, B1c45, B146, and R13e2) were sensitive to albusi...

Distribution and Genetic Diversity of Bacteriocin Gene Clusters in Rumen Microbial Genomes

“…Previous work reported the production of albusin B by R. albus 7 and R. albus 8, and two new R. albus strains whose genomes encode albusin-like bacteriocins were identified in this study. Albusin B was initially described to be a relatively hydrophilic, heatlabile, high-molecular-mass (Ͼ30 kDa) protein showing a narrow range of antibacterial activity (29). The putative class III bacteriocins in R. albus AD2013 and R. albus SY3 had identical amino acid sequences.…”

“…5). The amino acid sequence of albusin B from R. albus 7 has been described (29), and linocin was previously reported to be a putative class III bacteriocin produced by W. succinogenes DSM 1740, as determined using in silico analysis (30). Nonetheless, the analysis of the R. albus 7 genome revealed the genetic organization of the albusin B gene cluster, demonstrating the presence of an ABC transporter adjacent to albusin B and two ORFs encoding putative transposases related to the IS1595 and IS66 families.…”

Novel Lectin-Like Bacteriocins of Biocontrol Strain Pseudomonas fluorescens Pf-5

“…Chen and coworkers identified such an MMBL domain in a 32-kDa bacteriocin (albusin B or AlbB) produced by the ruminal bacterium Ruminococcus albus strain 7. They showed that albusin B is a potent growth inhibitor of Ruminococcus flavescens strains (11). Albusin B and LlpA BW11M1 are likely released from the bacteriocinogenic cells via a different route.…”

“…The mechanism by which this type of bacteriocin inhibits the growth of sensitive bacteria is still under investigation. Despite the presence of MMBL domains, LlpA and albusin B are unable to bind mannose (11,46).…”

“…Albusin B and LlpA BW11M1 are likely released from the bacteriocinogenic cells via a different route. LlpA BW11M1 secretion is signal peptide independent (46), while albusin B was shown to be produced as a prepeptide containing a 49-amino-acid signal peptide (11). The mechanism by which this type of bacteriocin inhibits the growth of sensitive bacteria is still under investigation.…”