Disruption of rcsB by a duplicated sequence in a curli-producing Escherichia coli O157:H7 results in differential gene expression in relation to biofilm formation, stress responses, and metabolism

Authors: Sharma, Vijay; Bayles, Darrell; Alt, David; Looft, Torey; Brunelle, Brian; Stasko, Judith

Submitted to: BMC Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/27/2017
Publication Date: 3/8/2017
Citation: Sharma, V.K., Bayles, D.O., Alt, D.P., Looft, T.P., Brunelle, B.W., Stasko, J.A. 2017. Disruption of rcsB by a duplicated sequence in a curli-producing Escherichia coli O157:H7 results in differential gene expression in relation to biofilm formation, stress responses, and metabolism. BMC Microbiology. 17(1):56. doi: 10.1186/s12866-017-0966-x.

Interpretive Summary: Escherichia coli O157:H7 (O157) causes sporadic outbreaks of diarrheal illnesses in humans through the consumption of undercooked ground beef, raw vegetables, and water contaminated with O157 bacteria. The major sources for the contamination of these foods and water are cattle feces or manure containing O157. Since O157 bacteria preferentially colonize the digestive system, colonized cattle can shed these bacteria in their feces, which pose major risk to the contamination of cattle carcasses that are processed into ground beef and other meat cuts. These feces are also a risk factor for the contamination of drinking water as well as run-off water from cattle production facilities posing the down-stream risk to the contamination of vegetables and other produce. The genetic mechanisms employed by O157 bacteria to colonize and persist in the digestive tract of cattle and in the external environment are not fully understood. New methods for determining the entire repertoire of genetic information expressed by bacterial samples are becoming increasingly valuable to identify genetic traits that specifically promote ability of a bacterial sample compared to another sample to cause infection in humans, colonize a susceptible host animal, and survive in the environment. By using one of these technologies, we determined the total genetic information expressed by a mutant O157 sample, which based on a few selected tests appeared to be better equipped for survival in the environment compared to the parent sample that it was originated from. By comparing the genetic information expressed in the mutant to that of the parent O157 sample, we identified a unique set of traits expressed in the mutant sample that could potentially enhance its survival in the environment and presumably in cattle. The practical field applications of having detailed information about the genetic traits unique to different O157 samples would be useful for developing strategies to mitigate carriage of these bacteria in the animal and survival in the environment.

Technical Abstract: Background: Escherichia coli O157:H7 (O157) strain 86-24, linked to a 1986 disease outbreak, displays biofilm- and curli-negative phenotypes that are correlated with the lack of Congo red (CR) binding and formation of white colonies (CR negative) on a CR negative containing medium. However, on a CR medium this strain produces red isolates capable of producing biofilm and curli fimbriae. Results: To identify genes controlling differential expression of curli fimbriae and biofilm formation, RNA-Seq profile of a CR plus isolate was compared to the CR negative parental isolate. Of the 242 genes showing differential expression in the CR plus isolate, 201 genes encoded proteins of known functions while 41 encoded hypothetical proteins. Among the genes with known functions, 149 were downregulated and 52 were upregulated in expression. Some of the upregulated genes were linked to biofilm formation through biosynthesis of curli fimbriae and flagella. The genes encoding transcriptional regulators, such as CsgD, QseB, YkgK, YdeH, Bdm, CspD, BssR, and FlhDC, which modulate biofilm formation, were significantly altered in their expression. Several genes of the envelope stress (cpxP), heat shock (rpoH, htpX, degP), oxidative stress (ahpC, katE), nutrient limitation stress (phoB negative phoR and pst) response pathways, and amino acid metabolism were downregulated in the CR positive isolate. Many genes mediating acid resistance and colanic acid biosynthesis, which influence biofilm formation directly or indirectly, were also down-regulated. Comparative genomics of CR positive and CR negative isolates revealed the presence of a short duplicated sequence in the rcsB gene of the CR positive isolate. The alignment of the amino acid sequences of RcsB of the two isolates showed truncation of RcsB in the CR positive isolate at the insertion site of the duplicated sequence. Complementation of CR positive isolate with rcsB of the CR negative parent restored parental phenotypes to the CR positive isolate. Conclusion: The results of this study indicate that RcsB is a global regulator affecting bacterial survival in growth-restrictive environments and conserving energy through upregulation of genes promoting biofilm formation while downregulating certain metabolic functions. The rcsB inactivation that gives rise to CR positive isolates may confer adaptive advantages on O157 that are important to understand for developing strategies to mitigate carriage of these bacteria in the animal and survival in the environment.