Implications of Adenylate Metabolism in Hygiene Assessment: A Review.

The assessment of a hygienic state or cleanliness of contact surfaces has significant implications for food and medical industries seeking to monitor sanitation and exert improved control over a host of operations affecting human health. Methods used to make such assessments commonly involve visual inspections, standard microbial plating practices, and the application of ATP-based assays. Visual methods for inspection of hygienic states are inherently subjective and limited in efficacy by the accuracy of human senses, the degree of task-specific work experience, and various sources of human bias. Standard microbial swabbing and plating techniques are limited in that they require hours or even days of incubation to generate results, with such steps as enrichment and colony outgrowth resulting in delays that are often incompatible with manufacturing or usage schedules. Rapid in conduct and considered more objective in operation than visual or tactile inspection techniques, swabbing surfaces using ATP-based assessments are relied on as routine, even standard, methods of hygienic assessment alone or in complement with microbial and visual inspection methods. Still, current ATP methods remain indirect methods of total hygiene assessment and have limitations that must be understood and considered if such methods are to be applied judiciously, especially under increasingly strict demands for the verification of hygiene state. Here, we present current methods of ATP-based bioluminescence assays and describe the limitations of such methods when applied to general food manufacturing or health care facilities.

RNase E-based degradosome modulates polyadenylation of mRNAs after Rho-independent transcription terminators in Escherichia coli.

Here we demonstrate that the RNase E-based degradosome is required for poly(A) polymerase I (PAP I)-dependent polyadenylation after Rho-independent transcription terminators for both mono- and polycistronic transcripts. Disruption of degradosome assembly in mutants lacking the polynucleotide phosphorylase (PNPase) binding domain led to a significant increase in the level of PNPase synthesized polynucleotide tails in the rpsJ and rpsM polycistronic transcripts and the lpp monocistronic transcript. The polynucleotide tails were mostly located within the coding sequences in the degradosome mutants compared to the wild type control where the majority of the PAP I synthesized poly(A) tails were after the Rho-independent transcription terminators. For the Rho terminated metNIQ operon, the tails for all three mRNAs were predominately polynucleotide and were located within the coding sequences in both wild type and degradosome mutant strains. Furthermore, by employing a pnp-R100D point mutant that encodes a catalytically inactive PNPase protein that still forms intact degradosomes, we show that a catalytically active PNPase is required for normal mRNA polyadenylation by PAP I. Our data suggest that polyadenylation requires a functional degradosome to maintain an equilibrium between free PNPase and the PAP I polyadenylation complex.