Dual-species biofilm of Listeria monocytogenes and Escherichia coli on stainless steel surface.

Listeria monocytogenes is a Gram-positive bacterium commonly associated with foodborne diseases. Due its ability to survive under adverse environmental conditions and to form biofilm, this bacterium is a major concern for the food industry, since it can compromise sanitation procedures and increase the risk of post-processing contamination. Little is known about the interaction between L. monocytogenes and Gram-negative bacteria on biofilm formation. Thus, in order to evaluate this interaction, Escherichia coli and L. monocytogenes were tested for their ability to form biofilms together or in monoculture. We also aimed to evaluate the ability of L. monocytogenes 1/2a and its isogenic mutant strain (ΔprfA ΔsigB) to form biofilm in the presence of E. coli. We assessed the importance of the virulence regulators, PrfA and σB, in this process since they are involved in many aspects of L. monocytogenes pathogenicity. Biofilm formation was assessed using stainless steel AISI 304 #4 slides immersed into brain heart infusion broth, reconstituted powder milk and E. coli preconditioned medium at 25 °C. Our results indicated that a higher amount of biofilm was formed by the wild type strain of L. monocytogenes than by its isogenic mutant, indicating that prfA and sigB are important for biofilm development, especially maturation under our experimental conditions. The presence of E. coli or its metabolites in preconditioned medium did not influence biofilm formation by L. monocytogenes. Our results confirm the possibility of concomitant biofilm formation by L. monocytogenes and E. coli, two bacteria of major significance in the food industry.

Protein synthesis factors (RF1, RF2, RF3, RRF, and tmRNA) and peptidyl-tRNA hydrolase rescue stalled ribosomes at sense codons.

During translation, ribosomes stall on mRNA when the aminoacyl-tRNA to be read is not readily available. The stalled ribosomes are deleterious to the cell and should be rescued to maintain its viability. To investigate the contribution of some of the cellular translation factors on ribosome rescuing, we provoked stalling at AGA codons in mutants that affected the factors and then analyzed the accumulation of oligopeptidyl (peptides of up to 6 amino acid residues, oligopep-)-tRNA or polypeptidyl (peptides of more than 300 amino acids in length, polypep-)-tRNA associated with ribosomes. Stalling was achieved by starvation for aminoacyl-tRNA(Arg4) upon induced expression of engineered lacZ (ß-galactosidase) reporter gene harboring contiguous AGA codons close to the initiation codon or at internal codon positions together with minigene ATGAGATAA accompanied by reduced peptidyl-tRNA hydrolase (Pth). Our results showed accumulations of peptidyl-tRNA associated with ribosomes in mutants for release factors (RF1, RF2, and RF3), ribosome recycling factor (RRF), Pth, and transfer-messenger RNA (tmRNA), implying that each of these factors cooperate in rescuing stalled ribosomes. The role of these factors in ribosome releasing from the stalled complex may vary depending on the length of the peptide in the peptidyl-tRNA. RF3 and RRF rescue stalled ribosomes by "drop-off" of peptidyl-tRNA, while RF1, RF2 (in the absence of termination codon), or Pth may rescue by hydrolyzing the associated peptidyl-tRNA. This is followed by the disassembly of the ribosomal complex of tRNA and mRNA by RRF and elongation factor G.

A fluorescent mutant of the NM domain of the yeast prion Sup35 provides insight into fibril formation and stability.

The Sup35 protein of Saccharomyces cerevisiae forms a prion that generates the [PSI(+)] phenotype. Its NM region governs prion status, forming self-seeding amyloid fibers in vivo and in vitro. A tryptophan mutant of Sup35 (NM(F117W)) was used to probe its aggregation. Four indicators of aggregation, Trp 117 maximum emission, Trp polarization, thio-T binding, and light scattering increase, revealed faster aggregation at 4 degrees C than at 25 degrees C, and all indicators changed in a concerted fashion at the former temperature. Curiously, at 25 degrees C the changes were not synchronized; the first two indicators, which reflect nucleation, changed more quickly than the last two, which reflect fibril formation. These results suggest that nucleation is insensitive to temperature, whereas fibril extension is temperature dependent. As expected, aggregation is accelerated when a small fraction (5%) of the nuclei produced at 4 or 25 degrees C are added to a suspension containing the soluble NM domain, although these nuclei do not seem to propagate any structural information to the growing fibrils. Fibrils grown at 4 degrees C were less stable in GdmCl than those grown at higher temperature. However, they were both resistant to high pressure; in fact, both sets of fibrils responded to high pressure by adopting an altered conformation with a higher capacity for thio-T binding. From these data, we calculated the change in volume and free energy associated with this conformational change. AFM revealed that the fibrils grown at 4 degrees C were statistically smaller than those grown at 25 degrees C. In conclusion, the introduction of Trp 117 allowed us to more carefully dissect the effects of temperature on the aggregation of the Sup35 NM domain.