Modeling and validation of autoinducer-mediated bacterial gene expression in microfluidic environments.

Biosensors exploiting communication within genetically engineered bacteria are becoming increasingly important for monitoring environmental changes. Currently, there are a variety of mathematical models for understanding and predicting how genetically engineered bacteria respond to molecular stimuli in these environments, but as sensors have miniaturized towards microfluidics and are subjected to complex time-varying inputs, the shortcomings of these models have become apparent. The effects of microfluidic environments such as low oxygen concentration, increased biofilm encapsulation, diffusion limited molecular distribution, and higher population densities strongly affect rate constants for gene expression not accounted for in previous models. We report a mathematical model that accurately predicts the biological response of the autoinducer N-acyl homoserine lactone-mediated green fluorescent protein expression in reporter bacteria in microfluidic environments by accommodating these rate constants. This generalized mass action model considers a chain of biomolecular events from input autoinducer chemical to fluorescent protein expression through a series of six chemical species. We have validated this model against experimental data from our own apparatus as well as prior published experimental results. Results indicate accurate prediction of dynamics (e.g., 14% peak time error from a pulse input) and with reduced mean-squared error with pulse or step inputs for a range of concentrations (10 µM-30 µM). This model can help advance the design of genetically engineered bacteria sensors and molecular communication devices.

Non-coding sRNAs regulate virulence in the bacterial pathogen Vibrio cholerae.

Vibrio cholerae is the waterborne bacterium responsible for worldwide outbreaks of the acute, potentially fatal cholera diarrhea. The primary factors this human pathogen uses to cause the disease are controlled by a complex regulatory program linking extracellular signaling inputs to changes in expression of several critical virulence genes. Recently it has been uncovered that many non-coding regulatory sRNAs are important components of the V. cholerae virulence regulon. Most of these sRNAs appear to require the RNA-binding protein, Hfq, to interact with and alter the expression of target genes, while a few sRNAs appear to function by an Hfq-independent mechanism. Direct base-pairing between the sRNAs and putative target mRNAs has been shown in a few cases but the extent of each sRNAs regulon is not fully known. Genetic and biochemical methods, coupled with computational and genomics approaches, are being used to validate known sRNAs and also to identify many additional putative sRNAs that may play a role in the pathogenic lifestyle of V. cholerae.

The Vibrio cholerae quorum sensing response is mediated by Hfq-dependent sRNA/mRNA base pairing interactions.

Vibrio cholerae quorum sensing controls expression of four redundant sRNAs, Qrr1-4. The Qrr sRNAs are predicted to alter the translation of several mRNAs, including, hapR, which encodes a transcription factor that controls genes for virulence factors, biofilm formation, protease production and DNA uptake. Each Qrr contains a 21 nucleotide region absolutely conserved among pathogenic Vibrios, and predicted to base pair with mRNA targets, like hapR, aided by the RNA chaperone Hfq. This molecular mechanism was not experimentally tested previously, and we provide here both in vivo and in vitro evidence to validate this model. In Escherichia coli, Qrr expression repressed a HapR-GFP translational fusion, and a specific nucleotide substitution in the 21 nucleotide region eliminated HapR control, while a compensatory mutation in hapR restored it. In V. cholerae, the identical mutations also deregulated HapR-dependent gene expression and corresponding QS phenotypes by altering HapR protein levels. We calculated in vitro binding affinities of a Qrr/hapR complex and show that Hfq stabilizes complex formation. Finally, the Qrr mutation with in vivo defects also prevented Qrr/hapR binding, while the compensatory hapR mutation restored binding. These results demonstrate that the V. cholerae QS response is mediated by base pairing interactions between Qrr sRNAs and hapR mRNA.

The spontaneous appearance rate of the yeast prion [PSI+] and its implications for the evolution of the evolvability properties of the [PSI+] system.

Epigenetically inherited aggregates of the yeast prion [PSI+] cause genomewide readthrough translation that sometimes increases evolvability in certain harsh environments. The effects of natural selection on modifiers of [PSI+] appearance have been the subject of much debate. It seems likely that [PSI+] would be at least mildly deleterious in most environments, but this may be counteracted by its evolvability properties on rare occasions. Indirect selection on modifiers of [PSI+] is predicted to depend primarily on the spontaneous [PSI+] appearance rate, but this critical parameter has not previously been adequately measured. Here we measure this epimutation rate accurately and precisely as 5.8 x 10(-7) per generation, using a fluctuation test. We also determine that genetic "mimics" of [PSI+] account for up to 80% of all phenotypes involving general nonsense suppression. Using previously developed mathematical models, we can now infer that even in the absence of opportunities for adaptation, modifiers of [PSI+] are only weakly deleterious relative to genetic drift. If we assume that the spontaneous [PSI+] appearance rate is at its evolutionary optimum, then opportunities for adaptation are inferred to be rare, such that the [PSI+] system is favored only very weakly overall. But when we account for the observed increase in the [PSI+] appearance rate in response to stress, we infer much higher overall selection in favor of [PSI+] modifiers, suggesting that [PSI+]-forming ability may be a consequence of selection for evolvability.

Requirements of Hsp104p activity and Sis1p binding for propagation of the [RNQ(+)] prion.

The formation and maintenance of prions in the yeast Saccharomyces cerevisiae is highly regulated by the cellular chaperone machinery. The most important player in this regulation is Hsp104p, which is required for the maintenance of all known prions. The requirements for other chaperones, such as members of the Hsp40 or Hsp70 families, vary with each individual prion. [RNQ(+)] cells do not have a phenotype that is amenable to genetic screens to identify cellular factors important in prion propagation. Therefore, we used a chimeric construct that reports the [RNQ(+)] status of cells to perform a screen for mutants that are unable to maintain [RNQ(+)]. We found eight separate mutations in Hsp104p that caused [RNQ(+)] cells to become [rnq(-)]. These mutations also caused the loss of the [PSI(+)] prion. The expression of one of these mutants, Hsp104p-E190K, showed differential loss of the [RNQ(+)] and [PSI(+)] prions in the presence of wild type Hsp104p. Hsp104p-E190K inefficiently propagated [RNQ(+)] and was unable to maintain [PSI(+)]. The mutant was unable to act on other in vivo substrates, as strains carrying it were not thermotolerant. Purified recombinant Hsp104p-E190K showed a reduced level of ATP hydrolysis as compared to wild type protein. This is likely the cause of both prion loss and lack of in vivo function. Furthermore, it suggests that [RNQ(+)] requires less Hsp104p activity to maintain transmissible protein aggregates than Sup35p. Additionally, we show that the L94A mutation in Rnq1p, which reduces its interaction with Sis1p, prevents Rnq1p from maintaining a prion and inducing [PSI(+)].

Heterologous prion interactions are altered by mutations in the prion protein Rnq1p.

Prions in the yeast Saccharomyces cerevisiae show a surprising degree of interdependence. Specifically, the rate of appearance of the [PSI+] prion, which is thought to be an important mechanism to respond to changing environmental conditions, is greatly increased by another prion, [RNQ+]. While the domains of the Rnq1 protein important for formation of the [RNQ+] prion have been defined, the specific residues required remain unknown. Furthermore, residues in Rnq1p that mediate the interaction between [PSI+] and [RNQ+] are unknown. To identify residues important for prion protein interactions, we created a mutant library of Rnq1p clones in the context of a chimera that serves as proxy for [RNQ+] aggregates. Several of the mutant Rnq1p proteins showed structural differences in the aggregates they formed, as revealed by semi-denaturing detergent agarose gel electrophoresis. Additionally, several of the mutants showed a striking defect in the ability to promote [PSI+] induction. These data indicate that the mutants formed strain variants of [RNQ+]. By dissecting the mutations in the isolated clones, we found five single mutations that caused [PSI+] induction defects, S223P, F184S, Q239R, N297S, and Q298R. These are the first specific mutations characterized in Rnq1p that alter [PSI+] induction. Additionally, we have identified a region important for the propagation of certain strain variants of [RNQ+]. Deletion of this region (amino acids 284-317) affected propagation of the high variant but not medium or low [RNQ+] strain variants. Furthermore, when the low [RNQ+] strain variant was propagated by Delta284-317, [PSI+] induction was greatly increased. These data suggest that this region is important in defining the structure of the [RNQ+] strain variants. These data are consistent with a model of [PSI+] induction caused by physical interactions between Rnq1p and Sup35p.

IcmS-dependent translocation of SdeA into macrophages by the Legionella pneumophila type IV secretion system.

Legionella pneumophila replicates inside alveolar macrophages and causes an acute, potentially fatal pneumonia called Legionnaires' disease. The ability of this bacterium to grow inside of macrophages is dependent on the presence of a functional dot/icm type IV secretion system (T4SS). Proteins secreted by the Dot/Icm T4SS are presumed to alter the host endocytic pathway, allowing L. pneumophila to establish a replicative niche within the host cell. Here we show that a member of the SidE family of proteins interacts with IcmS and is required for full virulence in the protozoan host Acanthamoeba castellanii. Using immunofluorescence microscopy and adenylate cyclase fusions, we show that SdeA is secreted into host cells by L. pneumophila in an IcmS-dependent manner. The SidE-like proteins are secreted very early during macrophage infection, suggesting that they are important in the initial formation of the replicative phagosome. Secreted SidE family members show a similar localization to other Dot/Icm substrates, specifically, to the poles of the replicative phagosome. This common localization of secreted substrates of the Dot/Icm system may indicate the formation of a multiprotein complex on the cytoplasmic face of the replicative phagosome.

Noninvasive bioluminescence imaging of herpes simplex virus type 1 infection and therapy in living mice.

Mouse models of herpes simplex virus type 1 (HSV-1) infection provide significant insights into viral and host genes that regulate disease pathogenesis, but conventional methods to determine the full extent of viral spread and replication typically require the sacrifice of infected animals. To develop a noninvasive method for detecting HSV-1 in living mice, we used a strain KOS HSV-1 recombinant that expresses firefly (Photinus pyralis) and Renilla (Renilla reniformis) luciferase reporter proteins and monitored infection with a cooled charge-coupled device camera. Viral infection in mouse footpads, peritoneal cavity, brain, and eyes could be detected by bioluminescence imaging of firefly luciferase. The activity of Renilla luciferase could be imaged after direct administration of substrate to infected eyes but not following the systemic delivery of substrate. The magnitude of bioluminescence from firefly luciferase measured in vivo correlated directly with input titers of recombinant virus used for infection. Treatment of infected mice with valacyclovir, a potent inhibitor of HSV-1 replication, produced dose-dependent decreases in firefly luciferase activity that correlated with changes in viral titers. These data demonstrate that bioluminescence imaging can be used for noninvasive, real-time monitoring of HSV-1 infection and therapy in living mice.