Pratt et al., 2012

Rod-shaped bacteria form filaments with multiple nucleoids when division, but not growth, is disrupted. Reducing the water activity (a_w) of food items is one method of inhibiting bacterial growth. Salmonella enterica form smooth, aseptate filaments with multiple nucleoids by an unknown mechanism when cultured on reduced-a_w media (achieved by supplementing media with sodium chloride). Multi-chromosomal filaments are viable and form single colonies on agar plates even though they are actually comprised of multiple cells (10-50). Introduction of filaments into favorable culture conditions leads to septa formation and separation of the filament into individual cells with a shorter lag phase and more rapid exponential growth phase than non-filamentous control cells. Thus, samples containing filamentous salmonellae can yield misleading numbers of CFU and presents challenges to retrospective determinations of infectious dose and risk assessments. We sought to characterize the underlying mechanisms of osmotic-induced filament formation. Protein synthesis and DNA replication were similar in filament and control cells when suspensions were standardized by optical density (600 nm). Furthermore, penicillin-binding proteins in the outer membrane of salmonellae were functional and capable of binding a fluorescent derivative of penicillin V (BOCILLIN-FL) in vitro. These data indicate that osmotic-induced filamentation is not a result of disregulation of DNA or protein synthesis, or protein folding. The level of the penicillin-binding protein 2 as determined with BOCILLIN-FL was greater in filaments than control cells, suggesting it has a role in filament formation. Filaments also contained 3-fold more ATP than did control cells in standardized cell suspensions. The significance of this finding is unclear, though, it may explain how filaments are able to divide and septate within eight hours in 0.2× Luria-Bertani broth at 23°C, while control cells do not. Based upon this finding a method was developed to enumerate the number of individual cells comprised with a filament suspension—considered here as multiple “viable units”—by plate-count. Finally, membrane proteins that increased or decreased during osmotic-induced filament formation were identified. These proteins are being investigated for possible roles in filament formation.

Park et al., 2011

E. coli O157 :H7 produces Shiga-like toxin and is a major threat to public health worldwide. The tolerance of this bacterium to various external stresses are exceptional and mirror its high levels of genomic plasticity. The genome of E. coli O157:H7 EDL933 contains 18 multigenic prophage regions. The genome diversity of O157 strains is largely due to genetic rearrangements mediated by prophage. Cattle are considered the primary reservoir for Escherichia coli O157 and are often a source of new variants. Heterogeneity of REDP (restriction endonuclease digestion profiles) among bovine-derived E. coli O157 strains implies the presence of on-going genetic drift. To further understand the molecular evolution of E. coli O157, 18 bovine isolates representing different REDP groups from two Wisconsin dairy farms (R and X) were selected based on the prevalence characteristics identified in the previous studies. Molecular characterizations of the O157 isolates were conducted to determine polymorphic patterns of prophage regions, subtypes and expression of Shiga toxin gene(s), and bacteriophage production (induced lysogens). The results showed that the genetic histories of the E. coli O157 on farms R and X were strikingly different. Farm R was predominated by a single genetic lineage (R^P) that possesses an unusual genetic feature related to prophage. Its prophages are highly polymorphic and likely defective. In particular, Shiga toxin-converting prophage BP-933W contained extensive gene deletion and encoded a variant type of Shiga toxin 2 rather than a prototypic stx2 A subtype. It concomitantly abolished Stx2 expression and lytic production of BP-933W. R^P lineage persisted on farm R for 8 years with more than 99% prevalence. In contrast, the genetic history of E. coli O157 on farm X was comprised of 6 distinct genetic groups. These groups are heterogeneous in terms of the prophage-related characteristics. Specifically, strains similar to R^P lineage, Shiga toxin producing EDL933-like strains, and a variant producing an alternative prophage CP-933U contributed to genetic dynamics of O157 on farm X.

Comparative molecular analysis of a longitudinal collection of Escherichia coli O157:H7 isolates from two different farms provided evidence that molecular evolution of this pathogen is niche-specific and largely involves prophage-mediated genetic drifts which is not only limited to chromosomal polymorphisms but extends to the induction of lysogens as well as the expression of virulence factors carried by prophages.

• Microbiology 303:Biology of Microorganisms

• Food Research Institute
• Professor, Department of Animal Sciences

• Molecular and Environmental Toxicology Center