Experimental and Genomic Evaluation of the Oestrogen Degrading Bacterium Rhodococcus equi ATCC13557.

Rhodococcus equi ATCC13557 was selected as a model organism to study oestrogen degradation based on its previous ability to degrade 17α-ethinylestradiol (EE2). Biodegradation experiments revealed that R. equi ATCC13557 was unable to metabolise EE2. However, it was able to metabolise E2 with the major metabolite being E1 with no further degradation of E1. However, the conversion of E2 into E1 was incomplete, with 11.2 and 50.6% of E2 degraded in mixed (E1-E2-EE2) and E2-only conditions, respectively. Therefore, the metabolic pathway of E2 degradation by R. equi ATCC13557 may have two possible pathways. The genome of R. equi ATCC13557 was sequenced, assembled, and mapped for the first time. The genome analysis allowed the identification of genes possibly responsible for the observed biodegradation characteristics of R. equi ATCC13557. Several genes within R. equi ATCC13557 are similar, but not identical in sequence, to those identified within the genomes of other oestrogen degrading bacteria, including Pseudomonas putida strain SJTE-1 and Sphingomonas strain KC8. Homologous gene sequences coding for enzymes potentially involved in oestrogen degradation, most commonly a cytochrome P450 monooxygenase (oecB), extradiol dioxygenase (oecC), and 17ß-hydroxysteroid dehydrogenase (oecA), were identified within the genome of R. equi ATCC13557. These searches also revealed a gene cluster potentially coding for enzymes involved in steroid/oestrogen degradation; 3-carboxyethylcatechol 2,3-dioxygenase, 2-hydroxymuconic semialdehyde hydrolase, 3-alpha-(or 20-beta)-hydroxysteroid dehydrogenase, 3-(3-hydroxy-phenyl)propionate hydroxylase, cytochrome P450 monooxygenase, and 3-oxosteroid 1-dehydrogenase. Further, the searches revealed steroid hormone metabolism gene clusters from the 9, 10-seco pathway, therefore R. equi ATCC13557 also has the potential to metabolise other steroid hormones such as cholesterol.

Driving the expression of the Salmonella enterica sv Typhimurium flagellum using flhDC from Escherichia coli results in key regulatory and cellular differences.

The flagellar systems of Escherichia coli and Salmonella enterica exhibit a significant level of genetic and functional synteny. Both systems are controlled by the flagellar specific master regulator FlhD4C2. Since the early days of genetic analyses of flagellar systems it has been known that E. coli flhDC can complement a ∆flhDC mutant in S. enterica. The genomic revolution has identified how genetic changes to transcription factors and/or DNA binding sites can impact the phenotypic outcome across related species. We were therefore interested in asking: using modern tools to interrogate flagellar gene expression and assembly, what would the impact be of replacing the flhDC coding sequences in S. enterica for the E. coli genes at the flhDC S. entercia chromosomal locus? We show that even though all strains created are motile, flagellar gene expression is measurably lower when flhDCEC are present. These changes can be attributed to the impact of FlhD4C2 DNA recognition and the protein-protein interactions required to generate a stable FlhD4C2 complex. Furthermore, our data suggests that in E. coli the internal flagellar FliT regulatory feedback loop has a marked difference with respect to output of the flagellar systems. We argue due diligence is required in making assumptions based on heterologous expression of regulators and that even systems showing significant synteny may not behave in exactly the same manner.

Growth rate control of flagellar assembly in Escherichia coli strain RP437.

The flagellum is a rotary motor that enables bacteria to swim in liquids and swarm over surfaces. Numerous global regulators control flagellar assembly in response to cellular and environmental factors. Previous studies have also shown that flagellar assembly is affected by the growth-rate of the cell. However, a systematic study has not yet been described under controlled growth conditions. Here, we investigated the effect of growth rate on flagellar assembly in Escherichia coli using steady-state chemostat cultures where we could precisely control the cell growth-rate. Our results demonstrate that flagellar abundance correlates with growth rate, where faster growing cells produce more flagella. They also demonstrate that this growth-rate dependent control occurs through the expression of the flagellar master regulator, FlhD4C2. Collectively, our results demonstrate that motility is intimately coupled to the growth-rate of the cell.

A nutrient-tunable bistable switch controls motility in Salmonella enterica serovar Typhimurium.

UNLABELLED: Many bacteria are motile only when nutrients are scarce. In contrast, Salmonella enterica serovar Typhimurium is motile only when nutrients are plentiful, suggesting that this bacterium uses motility for purposes other than foraging, most likely for host colonization. In this study, we investigated how nutrients affect motility in S. enterica and found that they tune the fraction of motile cells. In particular, we observed coexisting populations of motile and nonmotile cells, with the distribution being determined by the concentration of nutrients in the growth medium. Interestingly, S. enterica responds not to a single nutrient but apparently to a complex mixture of them. Using a combination of experimentation and mathematical modeling, we investigated the mechanism governing this behavior and found that it results from two antagonizing regulatory proteins, FliZ and YdiV. We also found that a positive feedback loop involving the alternate sigma factor FliA is required, although its role appears solely to amplify FliZ expression. We further demonstrate that the response is bistable: that is, genetically identical cells can exhibit different phenotypes under identical growth conditions. Together, these results uncover a new facet of the regulation of the flagellar genes in S. enterica and further demonstrate how bacteria employ phenotypic diversity as a general mechanism for adapting to change in their environment. IMPORTANCE: Many bacteria employ flagella for motility. These bacteria are often not constitutively motile but become so only in response to specific environmental cues. The most common is nutrient starvation. Interestingly, in Salmonella enterica serovar Typhimurium, nutrients enhance the expression of flagella, suggesting that motility is used for purposes other than foraging. In this work, we investigated how nutrients affect motility in S. enterica and found that nutrients tune the fraction of motile cells within a population. Using both experimental and mathematical analysis, we determined the mechanism governing this tunable response. We further demonstrated that the response is bistable: that is, genetically identical cells can exhibit different phenotypes under identical growth conditions. These results reveal a new facet of motility in S. enterica and demonstrate that nutrients determine not only where these bacteria swim but also the fraction of them that do so.