Bacterial protein secretion — a target for new antibiotics?

AbstractThe heavy use of antibiotics over recent decades has resulted in widespread resistance of bacteria to many drugs. Overcoming resistance requires new approaches to antibiotic development, including the exploitation of new targets in the bacterial cell. Protein secretion is essential for bacterial cell growth and virulence, so it could be a suitable target for new therapeutic agents

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

A comprehensive set of plasmids for vanillate- and xylose-inducible gene expression in Caulobacter crescentus

Caulobacter crescentus is widely used as a powerful model system for the study of prokaryotic cell biology and development. Analysis of this organism is complicated by a limited selection of tools for genetic manipulation and inducible gene expression. This study reports the identification and functional characterization of a vanillate-regulated promoter (Pvan) which meets all requirements for application as a multi-purpose expression system in Caulobacter, thus complementing the established xylose-inducible system (Pxyl). Furthermore, we introduce a newly constructed set of integrating and replicating shuttle vectors that considerably facilitate cell biological and physiological studies in Caulobacter. Based on different narrow and broad-host range replicons, they offer a wide choice of promoters, resistance genes, and fusion partners for the construction of fluorescently or affinity-tagged proteins. Since many of these constructs are also suitable for use in other bacteria, this work provides a comprehensive collection of tools that will enrich many areas of microbiological research

Intellectual achievement in pursuit of true belief

The practice of inquiry, in which we seek and pursue true beliefs by forming justified beliefs, is important to us. This thesis will address two questions concerning the significance of this practice. These are the question of what explains our preference for this particular belief-forming practice, and whether this value can be explained by the value of true belief alone. To answer these questions I will examme a variety of our intuitive commitments to particular values, assuming their general accuracy. I will use an inference from the goal of a practice to the value of a practice, an inference based on the assumption that when we pursue something it is valuable. I will discuss our intuitive commitments to the value of justification. I will also rely on the implications of the presence of pride and admiration in relation to the outcome of an inquiry (especially in situations where a belief is difficult to form). By using this methodology, I will argue for three sources of value that explain the unique significance of the value of inquiry. The first is the value of its unique role in our being able to form reliably true beliefs. Second, I will argue for Wayne D. Riggs' account of epistemic credit; Riggs defends the value of our being responsible for true beliefs, they are our achievements. Third, I will argue for an additional the value of delivering a skilful epistemic performance, another kind of achievement. I will show that although the value of true belief plays a role in explaining some of the values, the third value for inquiry is independent of the value of true belief. This means that there are intellectual rewards, which can be gained from this practice, that extend beyond the value of true belief.KMBT_363Adobe Acrobat 9.54 Paper Capture Plug-i

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 functions of DNA methylation by CcrM in Caulobacter crescentus: a global approach

DNA methylation is involved in a diversity of processes in bacteria, including maintenance of genome integrity and regulation of gene expression. Here, using Caulobacter crescentus as a model, we exploit genome-wide experimental methods to uncover the functions of CcrM, a DNA methyltransferase conserved in most Alphaproteobacteria. Using single molecule sequencing, we provide evidence that most CcrM target motifs (GANTC) switch from a fully methylated to a hemi-methylated state when they are replicated, and back to a fully methylated state at the onset of cell division. We show that DNA methylation by CcrM is not required for the control of the initiation of chromosome replication or for DNA mismatch repair. By contrast, our transcriptome analysis shows that >10% of the genes are misexpressed in cells lacking or constitutively over-expressing CcrM. Strikingly, GANTC methylation is needed for the efficient transcription of dozens of genes that are essential for cell cycle progression, in particular for DNA metabolism and cell division. Many of them are controlled by promoters methylated by CcrM and co-regulated by other global cell cycle regulators, demonstrating an extensive cross talk between DNA methylation and the complex regulatory network that controls the cell cycle of C. crescentus and, presumably, of many other Alphaproteobacteri

Identification of the fliI and fliJ Components of the Caulobacter Flagellar Type III Protein Secretion System

Caulobacter crescentus is motile by virtue of a polar flagellum assembled during the predivisional stage of the cell cycle. Three mutant strains in which flagellar assembly was blocked at an early stage were isolated. The mutations in these strains mapped to an operon of two genes, fliI and fliJ, both of which are necessary for motility. fliI encodes a 50-kDa polypeptide whose sequence is closely related to that of the Salmonella typhimurium FliI protein, an ATPase thought to energize the export of flagellar subunits across the cytoplasmic membrane through a type III protein secretion system. fliJ encodes a 16-kDa hydrophilic protein of unknown function. Epistasis experiments demonstrated that the fliIJ operon is located in class II of the C. crescentus flagellar regulatory hierarchy, suggesting that the gene products act at an early stage in flagellar assembly. The expression of fliIJ is induced midway through the cell cycle, coincident with other class II operons, but the FliI protein remains present throughout the cell cycle. Subcellular fractionation showed that FliI is present both in the cytoplasm and in association with the membrane. Mutational analysis of FliI showed that two highly conserved amino acid residues in a bipartite ATP binding motif are necessary for flagellar assembly

Expansive components in H II regions

We study the presence of low intensity high velocity components, which we have termed wing features in the integrated Halpha emission line profiles of the HII region populations of the spiral barred galaxies NGC 1530, NGC 3359 and NGC 6951. We find that more than a third of the HII region line profiles in each galaxy show these components. The highest fraction is obtained in the galaxy whose line profiles show the best S:N, which suggests that wing features of this type may well exist in most, if not all, HII region line profiles. Applying selection criteria to the wing features, we obtain a sample of HII regions with clearly defined high velocity components in their profiles. Deconvolution of a representative sample of the line profiles eliminates any doubt that the wing features could possibly be due to instrumental effects. We present an analysis of the high velocity low intensity features fitting them with Gaussian functions; the emission measures, central velocities and velocity dispersions for the red and blue features take similar values. We interpret the features as signatures of expanding shells inside the HII regions. Up to a shell radius of R(shell)~0.2R(reg), the stellar winds from the central ionizing stars appear to satisfy the energy and momentum requirements for the formation and driving the shell. Several examples of the most luminous HII regions show that the shells appear to have larger radii; in these cases additional mechanisms may well be needed to explain the kinetic energies and momenta of the shells.Comment: 16 pages, 9 figures, accepted for publication in A&

A Molecular Perspective on Ammonia Chemistry in Atmospheric Water Droplets

Small scale chemical processes define familiar atmospheric systems such as smog, weather, and clouds. Dr. Joseph Francisco’s work uses computational chemistry to provide molecular level insights into the small scale processes that inform our understanding of the larger scale systems. We present two computational studies exploring ammonia transport and ion-pair formation at the air-water interface. These examples offer molecular perspectives on particle nucleation and ammonia cycling