Michael S. Allen

Using noise to probe and characterize gene circuits

Stochastic fluctuations (or “noise”) in the single-cell populations of molecular species are shaped by the structure and biokinetic rates of the underlying gene circuit. The structure of the noise is summarized by its autocorrelation function. In this article, we introduce the noise regulatory vector as a generalized framework for making inferences concerning the structure and biokinetic rates of a gene circuit from its noise autocorrelation function. Although most previous studies have focused primarily on the magnitude component of the noise (given by the zero-lag autocorrelation function), our approach also considers the correlation component, which encodes additional information concerning the circuit. Theoretical analyses and simulations of various gene circuits show that the noise regulatory vector is characteristic of the composition of the circuit. Although a particular noise regulatory vector does not map uniquely to a single underlying circuit, it does suggest possible candidate circuits, while excluding others, thereby demonstrating the probative value of noise in gene circuit analysis.

Frequency domain analysis of noise in simple gene circuits

Recent advances in single cell methods have spurred progress in quantifying and analyzing stochastic fluctuations, or noise, in genetic networks. Many of these studies have focused on identifying the sources of noise and quantifying its magnitude, and at the same time, paying less attention to the frequency content of the noise. We have developed a frequency domain approach to extract the information contained in the frequency content of the noise. In this article we review our work in this area and extend it to explicitly consider sources of extrinsic and intrinsic noise. First we review applications of the frequency domain approach to several simple circuits, including a constitutively expressed gene, a gene regulated by transitions in its operator state, and a negatively autoregulated gene. We then review our recent experimental study, in which time-lapse microscopy was used to measure noise in the expression of green fluorescent protein in individual cells. The results demonstrate how changes in rate constants within the gene circuit are reflected in the spectral content of the noise in a manner consistent with the predictions derived through frequency domain analysis. The experimental results confirm our earlier theoretical prediction that negative autoregulation not only reduces the magnitude of the noise but shifts its content out to higher frequency. Finally, we develop a frequency domain model of gene expression that explicitly accounts for extrinsic noise at the transcriptional and translational levels. We apply the model to interpret a shift in the autocorrelation function of green fluorescent protein induced by perturbations of the translational process as a shift in the frequency spectrum of extrinsic noise and a decrease in its weighting relative to intrinsic noise.

Preliminary Assessment of Microbiome Changes Following Blood-Feeding and Survivorship in the Amblyomma americanum Nymph-to-Adult Transition using Semiconductor Sequencing

The physiology of ticks supports a diverse community of non-pathogenic and pathogenic organisms. This study aims to initially characterize the microbial community present within colony-reared Amblyomma americanum using PCR of the variable region 5 of the 16S rRNA gene followed by semiconductor sequencing and classification of sequence data using the Ribosomal Database Project and MG-RAST analysis tools. Comparison of amplicon library datasets revealed changes in the microbiomes in newly engorged nymphs, newly-molted adults, and aged adults, as well as ticks exposed to different environmental conditions. These preliminary data support the concept that microbe survivorship and diversity are partially dependent upon environmental variables and the sequence of blood feeding, molting, and aging. The maintenance and/or emergence of pathogens in ticks may be dependent in part on temporal changes in the microbial community of the tick microbiome.

Noise in biological circuits

Noise biology focuses on the sources, processing, and biological consequences of the inherent stochastic fluctuations in molecular transitions or interactions that control cellular behavior. These fluctuations are especially pronounced in small systems where the magnitudes of the fluctuations approach or exceed the mean value of the molecular population. Noise biology is an essential component of nanomedicine where the communication of information is across a boundary that separates small synthetic and biological systems that are bound by their size to reside in environments of large fluctuations. Here we review the fundamentals of the computational, analytical, and experimental approaches to noise biology. We review results that show that the competition between the benefits of low noise and those of low population has resulted in the evolution of genetic system architectures that produce an uneven distribution of stochasticity across the molecular components of cells and, in some cases, use noise to drive biological function. We review the exact and approximate approaches to gene circuit noise analysis and simulation, and review many of the key experimental results obtained using flow cytometry and time-lapse fluorescent microscopy. In addition, we consider the probative value of noise with a discussion of using measured noise properties to elucidate the structure and function of the underlying gene circuit. We conclude with a discussion of the frontiers of and significant future challenges for noise biology.

Complete genome sequence of Thauera aminoaromatica strain MZ1T

Thauera aminoaromatica strain MZ1T, an isolate belonging to genus Thauera, of the family Rhodocyclaceae and the class the Betaproteobacteria, has been characterized for its ability to produce abundant exopolysaccharide and degrade various aromatic compounds with nitrate as an electron acceptor. These properties, if fully understood at the genome-sequence level, can aid in environmental processing of organic matter in anaerobic cycles by short-circuiting a central anaerobic metabolite, acetate, from microbiological conversion to methane, a critical greenhouse gas. Strain MZ1T is the first strain from the genus Thauera with a completely sequenced genome. The 4,496,212 bp chromosome and 78,374 bp plasmid contain 4,071 protein-coding and 71 RNA genes, and were sequenced as part of the DOE Community Sequencing Program CSP_776774.

A destabilized bacterial luciferase for dynamic gene expression studies

Fusions of genetic regulatory elements with reporter genes have long been used as tools for monitoring gene expression and have become a major component in synthetic gene circuit implementation. A major limitation of many of these systems is the relatively long half-life of the reporter protein(s), which prevents monitoring both the initiation and the termination of transcription in real-time. Furthermore, when used as components in synthetic gene circuits, the long time constants associated with reporter protein decay may significantly degrade circuit performance. In this study, short half-life variants of LuxA and LuxB from Photorhabdus luminescens were constructed in Escherichia coli by inclusion of an 11-amino acid carboxy-terminal tag that is recognized by endogenous tail-specific proteases. Results indicated that the addition of the C-terminal tag affected the functional half-life of the holoenzyme when the tag was added to luxA or to both luxA and luxB, but modification of luxB alone did not have a significant effect. In addition, it was also found that alteration of the terminal three amino acid residues of the carboxy-terminal tag fused to LuxA generated variants with half-lives of intermediate length in a manner similar to that reported for GFP. This report is the first instance of the C-terminal tagging approach for the regulation of protein half-life to be applied to an enzyme or monomer of a multi-subunit enzyme complex and will extend the utility of the bacterial luciferase reporter genes for the monitoring of dynamic changes in gene expression.

Frequency and Distribution of Rickettsiae, Borreliae, and Ehrlichiae Detected in Human-Parasitizing Ticks, Texas, USA

To describe the presence and distribution of tickborne bacteria and their vectors in Texas, USA, we screened ticks collected from humans during 2008–2014 for Rickettsia, Borrelia, and Ehrlichia spp. Thirteen tick species were identified, and 23% of ticks carried bacterial DNA from at least 1 of the 3 genera tested.

Flocculation-Related Gene Identification by Whole-Genome Sequencing of Thauera aminoaromatica MZ1T Floc-Defective Mutants

ABSTRACT Thauera aminoaromatica MZ1T, a floc-forming bacterium isolated from an industrial activated-sludge wastewater treatment plant, overproduces exopolysaccharide (EPS), leading to viscous bulking. This phenomenon results in poor sludge settling and dewatering during the clarification process. To identify genes responsible for bacterial flocculation, a whole-genome phenotypic-sequencing technique was applied. Genomic DNA of MZ1T flocculation-deficient mutants was subjected to massively parallel sequencing. The resultant high-quality reads were assembled and compared to the reference genome of the wild type (WT). We identified nine nonsynonymous mutations and one nonsense mutation putatively involved in EPS biosynthesis. Complementation of the nonsense mutation located in an EPS deacetylase gene restored the flocculating phenotype. The Fourier transform infrared (FTIR) spectra of EPS isolated from the wild type showed a reduced C=O peak of the N-acetyl group at 1,665 cm−1 compared to the spectra of MZ1T floc-deficient mutant EPS, suggesting that the WT EPS was partially deacetylated. Gene expression analysis also demonstrated that the putative deacetylase gene transcript increased before flocculation occurred. These data suggest that targeting deacetylation processes via direct chemical modification of EPS or enzyme inhibition may prove useful in combating viscous bulking in this and related bacteria.

Gene network shaping of inherent noise spectra

Recent work demonstrates that stochastic fluctuations in molecular populations have gene regulation consequences. Previous experiments focused on noise sources or noise propagation through gene networks by measuring noise magnitudes. However, in theoretical analysis we showed that noise frequency content is determined by the underlying gene circuits, leading to a mapping between gene circuit structure and the noise frequency range. An intriguing prediction was that negative autoregulation shifts noise to higher frequencies where it is more easily filtered out by gene networks, a property that may contribute to the prevalence (e.g. found in regulation of ~40% of E. coli genes) of autoregulation motifs. Here we measure noise frequency content in growing cultures of E. coli and verify the link between gene circuit structure and noise spectra by demonstrating the negative autoregulation-mediated spectral shift. We further demonstrate that noise spectral measurements provide mechanistic insights into gene regulation as perturbations of gene circuit parameters are discernible in the measured noise frequency ranges. These results suggest that noise spectral measurements could facilitate the discovery of novel regulatory relationships

Effects of temperature on bacterial microbiome composition in Ixodes scapularis ticks

Ixodes scapularis, the blacklegged deer tick, is the principal vector of Lyme disease in North America. Environmental factors are known to influence regional and seasonal incidence of Lyme disease and possibly the endemicity of the disease to the northeastern and upper mid‐western regions of the United States. With a goal to understand the impact of environmental temperature on microbial communities within the tick, we investigated the bacterial microbiome of colony‐reared I. scapularis ticks statically incubated at different temperatures (4, 20, 30, and 37°C) at a constant humidity in a controlled laboratory setting by comparison of sequenced amplicons of the bacterial 16S V4 rRNA gene to that of the untreated baseline controls. The microbiomes of colony‐reared I. scapularis males were distinct than that of females, which were entirely dominated by Rickettsia. In silico removal of Rickettsia sequences from female data revealed the underlying bacterial community, which is consistent in complexity with those seen among male ticks. The bacterial community composition of these ticks changes upon incubation at 30°C for a week and 37°C for more than 5 days. Moreover, the male ticks incubated at 30 and 37°C exhibited significantly different bacterial diversity compared to the initial baseline microbiome, and the change in bacterial diversity was dependent upon duration of exposure. Rickettsia‐free data revealed a significantly different bacterial diversity in female ticks incubated at 37°C compared to that of 4 and 20°C treatments. These results provide experimental evidence that environmental temperature can impact the tick bacterial microbiome in a laboratory setting.