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Gene expression is stochastic, and noise that arises from the stochastic nature of biochemical reactions propagates through active regulatory links. Thus, correlations in gene-expression noise can provide information about regulatory links. We present what to our knowledge is a new approach to measure and interpret such correlated fluctuations at the level of single microcolonies, which derive from single cells. We demonstrated this approach mathematically using stochastic modeling, and applied it to experimental time-lapse fluorescence microscopy data. Specifically, we investigated the relationships among LuxO, LuxR, and the small regulatory RNA qrr4 in the model quorum-sensing bacterium Vibrio harveyi. Our results show that LuxR positively regulates the qrr4 promoter. Under our conditions, we find that qrr regulation weakly depends on total LuxO levels and that LuxO autorepression is saturated. We also find evidence that the fluctuations in LuxO levels are dominated by intrinsic noise. We furthermore propose LuxO and LuxR interact at all autoinducer levels via an unknown mechanism. Of importance, our new method of evaluating correlations at the microcolony level is unaffected by partition noise at cell division. Moreover, the method is first-order accurate and requires less effort for data analysis than single-cell-based approaches. This new correlation approach can be applied to other systems to aid analysis of gene regulatory circuits., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Quorum-sensing is the mechanism by which bacteria communicate and synchronize group behaviors. Quantitative information on parameters such as the copy number of particular quorum-sensing proteins should contribute strongly to understanding how the quorum-sensing network functions. Here, we show that the copy number of the master regulator protein LuxR in Vibrio harveyi can be determined in vivo by exploiting small-number fluctuations of the protein distribution when cells undergo division. When a cell divides, both its volume and LuxR protein copy number, N, are partitioned with slight asymmetries. We measured the distribution functions describing the partitioning of the protein fluorescence and the cell volume. The fluorescence distribution is found to narrow systematically as the LuxR population increases, whereas the volume partitioning is unchanged. Analyzing these changes statistically, we determined that N = 80-135 dimers at low cell density and 575 dimers at high cell density. In addition, we measured the static distribution of LuxR over a large (3000) clonal population. Combining the static and time-lapse experiments, we determine the magnitude of the Fano factor of the distribution. This technique has broad applicability as a general in vivo technique for measuring protein copy number and burst size., (Copyright (c) 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

We investigated the binding interaction between the bacteriophage lambda-repressor CI and its target DNA using total internal reflection fluorescence microscopy. Large stepwise changes in the intensity of the red fluorescent protein fused to CI were observed as it associated with and dissociated from individually labeled single-molecule DNA targets. The stochastic association and dissociation were characterized by Poisson statistics. Dark and bright intervals were measured for thousands of individual events. The exponential distribution of the intervals allowed direct determination of the association and dissociation rate constants (k(a) and k(d), respectively). We resolved in detail how k(a) and k(d) varied as a function of three control parameters: the DNA length L, the CI dimer concentration, and the binding affinity. Our results show that although interactions with nonoperator DNA sequences are observable, CI binding to the operator site is not dependent on the length of flanking nonoperator DNA.