Bovine glycomacropeptide promotes the growth of Bifidobacterium longum ssp. infantis and modulates its gene expression.

Bovine milk glycomacropeptide (GMP) is derived from κ-casein, with exclusively o-linked glycosylation. Glycomacropeptide promoted the growth of Bifidobacterium longum ssp. infantis in a concentration-dependent manner, and this activity was lost following periodate treatment of the GMP (GMP-P), which disables biological recognition of the conjugated oligosaccharides. Transcriptional analysis of B. longum ssp. infantis following exposure to GMP revealed a substantial response to GMP relative to bacteria treated with GMP-P, with a greater number of differentially expressed transcripts and larger fold changes versus the control. Therefore, stimulation of B. longum ssp. infantis growth by GMP is intrinsically linked to the peptide's O-linked glycosylation. The pool of differentially expressed transcripts included 2 glycoside hydrolase (family 25) genes, which were substantially upregulated following exposure to GMP, but not GMP-P. These GH25 genes were present in duplicated genomic islands that also contained genes encoding fibronectin type III binding domain proteins and numerous phage-related proteins, all of which were also upregulated. Homologs of this genomic arrangement were present in other Bifidobacterium species, which suggest it may be a conserved domain for the utilization of glycosylated peptides. This study provides insights into the molecular basis for the prebiotic effect of bovine milk GMP on B. longum ssp. infantis.

Single-stage reconstruction of Achilles tendon injuries and distal lower extremity soft tissue defects with the reverse sural fasciocutaneous flap.

OBJECTIVE: Complex wounds of the lower extremity with concomitant Achilles tendon injury can be difficult to reconstruct. We favour the reverse sural artery fasciocutaneous flap because in a single step, flap elevation affords Achilles tendon exposure and adequate soft tissue for reconstruction. It also provides significant time and resource savings for both plastic and orthopaedic surgical teams. MATERIALS AND METHODS: Our case series involved four consecutive patients who presented with Achilles tendon injuries and concomitant complex soft tissue defects. The reverse sural artery flap was planned in conjunction with the orthopaedic service to facilitate their approach for Achilles tendon repair. Outcome was measured as flap survival, time for flap elevation and total operative time. RESULTS: Partial flap loss occurred in one patient. The Achilles repair was performed successfully in all cases. The mean time for flap elevation and Achilles exposure was 43 min (range, 37-52 min). Total operative time was 287 min (range, 211-347 min). CONCLUSION: The reverse sural artery fasciocutaneous flap is a durable, efficient option for simultaneous Achilles tendon reconstruction and wound coverage. Simple flap elevation provides necessary exposure of the Achilles tendon for repair while the flap itself provides ample soft tissue with a reliable blood supply. In our experience, the reverse sural artery fasciocutaneous flap affords a practical method to address two reconstructive challenges in a single procedure.

Exploitation of plasmid pMRC01 to direct transfer of mobilizable plasmids into commercial lactococcal starter strains.

Genetic analysis of the 60.2-kb lactococcal plasmid pMRC01 revealed a 19.6-kb region which includes putative genes for conjugal transfer of the plasmid and a sequence resembling an origin of transfer (oriT). This oriT-like sequence was amplified and cloned on a 312-bp segment into pCI372, allowing the resultant plasmid, pRH001, to be mobilized at a frequency of 3.4 x 10(-4) transconjugants/donor cell from an MG1363 (recA mutant) host containing pMRC01. All of the resultant chloramphenicol-resistant transconjugants contained both pRH001 and genetic determinants responsible for bacteriocin production and immunity of pMRC01. This result is expected, given that transconjugants lacking the lacticin 3147 immunity determinants (on pMRC01) would be killed by bacteriocin produced by the donor cells. Indeed, incorporation of proteinase K in the mating mixture resulted in the isolation of transformants, of which 47% were bacteriocin deficient. Using such an approach, the oriT-containing fragment was exploited to mobilize pRH001 alone to a number of lactococcal hosts. These results demonstrate that oriT of pMRC01 has the potential to be used in the development of mobilizable food-grade vectors for the genetic enhancement of lactococcal starter strains, some of which may be difficult to transform.

Potential of the enterocin regulatory system to control expression of heterologous genes in Enterococcus.

AIMS: To exploit the enterocin regulatory system for regulated expression of genes in Enterococcus. METHODS AND RESULTS: Production of some pediocin-like bacteriocins such as enterocin A in Enterococcus is regulated by a three-component system comprising a histidine kinase (entK ), a response regulator (entR) and an induction factor (entF ). Exposure to the induction factor results in the transcription of gene(s) under the control of the enterocin A promoter, including entA which encodes the bacteriocin. In an effort to exploit this system for expression of genes in Enterococcus, a number of vectors were constructed which contain the entA promoter followed by convenient cloning sites to introduce gene(s) of interest. These vectors were used in an enterococcal background which does not produce induction factor but does produce both the kinase and regulator proteins. The system was tested using the reporter genes ltnI (lacticin 3147 immunity) and gusA (beta-glucuronidase) under the control of the entA promoter. CONCLUSIONS: Upon addition of the induction factor, the beta-glucuronidase activity increased 20-fold when compared with uninduced cells. In addition, concentrations of as little as 0.2 nm synthetic EntF were sufficient to give maximal expression. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential benefit of having an expression system based on EntF is that gene expression can be finely controlled upon addition of low concentrations of a peptide that can easily be artificially synthesized.