Coordinate Cell Cycle Control of a Caulobacter DNA Methyltransferase and the Flagellar Genetic Hierarchy

The expression of the Caulobacter ccrM gene and the activity of its product, the M.Ccr II DNA methyltransferase, are limited to a discrete portion of the cell cycle (G. Zweiger, G. Marczynski, and L. Shapiro, J. Mol. Biol. 235:472-485, 1994). Temporal control of DNA methylation has been shown to be critical for normal development in the dimorphic Caulobacter life cycle. To understand the mechanism by which ccrM expression is regulated during the cell cycle, we have identified and characterized the ccrM promoter region. We have found that it belongs to an unusual promoter family used by several Caulobacter class II flagellar genes. The expression of these class II genes initiates assembly of the flagellum just prior to activation of the ccrM promoter in the predivisional cell. Mutational analysis of two M.Ccr II methylation sites located 3\u27 to the ccrM promoter suggests that methylation might influence the temporally controlled inactivation of ccrM transcription. An additional parallel between the ccrM and class II flagellar promoters is that their transcription responds to a cell cycle DNA replication checkpoint. We propose that a common regulatory system coordinates the expression of functionally diverse genes during the Caulobacter cell cycle

Caulobacter Lon protease has a critical role in cell-cvcle cbntrol of DNA I methylation

CcrM, an adenine DNA methyltransferase, is essential for viability in Caulobacter crescentus. The CcrM protein is present only in the predivisional stage of the cell cycle, resulting in cell-cycle-dependent variation of the DNA methylation state of the chromosome. The availability of CcrM is controlled in two ways: (1) the ccrM gene is transcribed only in the predivisional cell, and (2) the CcrM protein is rapidly degraded prior to cell division. We demonstrate here that CcrM is an important target of the Lon protease pathway in C. crescentus. In a lon null mutant, ccrM transcription is still temporally regulated, but the CcrM protein is present throughout the cell cycle because of a dramatic increase in its stability that results in a fully methylated chromosome throughout the cell cycle. Because the Lon protease is present throughout the cell cycle, it is likely that the level of CcrM in the cell is controlled by a dynamic balance between temporally varied transcription and constitutive degradation. We have shown previously that restriction of CcrM to the C. crescentus predivisional cell is essential for normal morphogenesis and progression through the cell cycle. Comparison of the lon null mutant strain with a strain whose DNA remains fully methylated as a result of constitutive expression of ccrM suggests that the effect of Lon on DNA methylation contributes to several developmental defects observed in the lon mutant. These defects include a frequent failure to complete cell division and loss of precise cell-cycle control of initiation of DNA replication. Other developmental abnormalities exhibited by the lon null mutant, such as the formation of abnormally long stalks, appear to be unrelated to altered chromosome methylation state. The Lon protease thus exhibits pleiotropic effects in C. crescentus growth and development

The Caulobacter crescentus DNA-(adenine-N6)-methyltransferase CcrM methylates DNA in a distributive manner

The specificity and processivity of DNA methyltransferases have important implications regarding their biological functions. We have investigated the sequence specificity of CcrM and show here that the enzyme has a high specificity for GANTC sites, with only minor preferences at the central position. It slightly prefers hemimethylated DNA, which represents the physiological substrate. In a previous work, CcrM was reported to be highly processive [Berdis et al. (1998) Proc. Natl Acad. Sci. USA 95: 2874–2879]. However upon review of this work, we identified a technical error in the setup of a crucial experiment in this publication, which prohibits making any statement about the processivity of CcrM. In this study, we performed a series of in vitro experiments to study CcrM processivity. We show that it distributively methylates six target sites on the pUC19 plasmid as well as two target sites located on a 129-mer DNA fragment both in unmethylated and hemimethylated state. Reaction quenching experiments confirmed the lack of processivity. We conclude that the original statement that CcrM is processive is no longer valid

Inscribing a discipline: tensions in the field of bioinformatics

Bioinformatics, the application of computer science to biological problems, is a central feature of post-genomic science which grew rapidly during the 1990s and 2000s. Post-genomic science is often high-throughput, involving the mass production of inscriptions [Latour and Woolgar (1986), Laboratory Life: the Construction of Scientific Facts. Princeton, NJ: Princeton University Press]. In order to render these mass inscriptions comprehensible, bioinformatic techniques are employed, with bioinformaticians producing what we call secondary inscriptions. However, despite bioinformaticians being highly skilled and credentialed scientists, the field struggles to develop disciplinary coherence. This paper describes two tensions militating against disciplinary coherence. The first arises from the fact that bioinformaticians as producers of secondary inscriptions are often institutionally dependent, subordinate even, to biologists. With bioinformatics positioned as service, it cannot determine its own boundaries but has them imposed from the outside. The second tension is a result of the interdisciplinary origin of bioinformatics – computer science and biology are disciplines with very different cultures, values and products. The paper uses interview data from two different UK projects to describe and examine these tensions by commenting on Calvert's [(2010) “Systems Biology, Interdisciplinarity and Disciplinary Identity.” In Collaboration in the New Life Sciences, edited by J. N. Parker, N. Vermeulen and B. Penders, 201–219. Farnham: Ashgate] notion of individual and collaborative interdisciplinarity and McNally's [(2008) “Sociomics: CESAGen Multidisciplinary Workshop on the Transformation of Knowledge Production in the Biosciences, and its Consequences.” Proteomics 8: 222–224] distinction between “black box optimists” and “black box pessimists.

Model-Based Deconvolution of Cell Cycle Time-Series Data Reveals Gene Expression Details at High Resolution

In both prokaryotic and eukaryotic cells, gene expression is regulated across the cell cycle to ensure “just-in-time” assembly of select cellular structures and molecular machines. However, present in all time-series gene expression measurements is variability that arises from both systematic error in the cell synchrony process and variance in the timing of cell division at the level of the single cell. Thus, gene or protein expression data collected from a population of synchronized cells is an inaccurate measure of what occurs in the average single-cell across a cell cycle. Here, we present a general computational method to extract “single-cell”-like information from population-level time-series expression data. This method removes the effects of 1) variance in growth rate and 2) variance in the physiological and developmental state of the cell. Moreover, this method represents an advance in the deconvolution of molecular expression data in its flexibility, minimal assumptions, and the use of a cross-validation analysis to determine the appropriate level of regularization. Applying our deconvolution algorithm to cell cycle gene expression data from the dimorphic bacterium Caulobacter crescentus, we recovered critical features of cell cycle regulation in essential genes, including ctrA and ftsZ, that were obscured in population-based measurements. In doing so, we highlight the problem with using population data alone to decipher cellular regulatory mechanisms and demonstrate how our deconvolution algorithm can be applied to produce a more realistic picture of temporal regulation in a cell

Global standards of Constitutional law : epistemology and methodology

Just as it led the philosophy of science to gravitate around scientific practice, the abandonment of all foundationalist aspirations has already begun making political philosophy into an attentive observer of the new ways in which constitutional law is practiced. Yet paradoxically, lawyers and legal scholars are not those who understand this the most clearly. Beyond analyzing the jurisprudence that has emerged from the expansion of constitutional justice, and taking into account the development of international and regional law, the ongoing globalization of constitutional law requires comparing the constitutional laws of individual nations. Following Waldron, the product of this new legal science can be considered as ius gentium. This legal science is not as well established as one might like to think. But it can be developed on the grounds of the practice that consists in ascertaining standards. As abstract types of best “practices” (and especially norms) of constitutional law from around the world, these are only a source of law in a substantive, not a formal, sense. They thus belong to what I should like to call a “second order legal positivity.” In this article I will undertake, both at a methodological and an epistemological level, the development of a model for ascertaining global standards of constitutional law

Temporal Controls of the Asymmetric Cell Division Cycle in Caulobacter crescentus

The asymmetric cell division cycle of Caulobacter crescentus is orchestrated by an elaborate gene-protein regulatory network, centered on three major control proteins, DnaA, GcrA and CtrA. The regulatory network is cast into a quantitative computational model to investigate in a systematic fashion how these three proteins control the relevant genetic, biochemical and physiological properties of proliferating bacteria. Different controls for both swarmer and stalked cell cycles are represented in the mathematical scheme. The model is validated against observed phenotypes of wild-type cells and relevant mutants, and it predicts the phenotypes of novel mutants and of known mutants under novel experimental conditions. Because the cell cycle control proteins of Caulobacter are conserved across many species of alpha-proteobacteria, the model we are proposing here may be applicable to other genera of importance to agriculture and medicine (e.g., Rhizobium, Brucella)