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

Der Translationsfaktor SelB

Die kotranslationelle Dekodierung des Kodons UGA als Selenocystein erfolgt durch eine spezifische tRNA (tRNASec), die von Seryl-tRNA Synthetase mit Serin beladen und anschließend von Selenocystein Synthase (SelA) zu Selenocysteyl- tRNASec umgesetzt wird. Selenophosphat, das als Selendonor für diese Reaktion dient, wird von Selenophosphat Synthetase (SelD) aus Selenid und ATP generiert. Der anschließende Transfer der beladenen tRNA zum Ribosom erfolgt durch den spezialisierten Elongationsfaktor SelB, dessen N-terminale Region Homologie zu EF-Tu zeigt und wie dieses Guanosin-Nukleotide und tRNA bindet. Der C-terminale Teil interagiert zusätzlich mit einer als SECIS-Element bezeichneten mRNA- Sekundärstruktur, die in Bakterien unmittelbar auf das für Selenocystein kodierende UGA-Triplett folgt und für dessen Rekodierung als Sinnkodon verantwortlich ist. Die vorliegende Arbeit beschäftigt sich mit den Mechanismen, die der Interaktion von SelB mit seinen Liganden sowie der Regulation der Selenocystein- Biosynthese durch SelB zu Grunde liegen. Im Einzelnen wurden dabei folgende Resultate erhalten: 1) Die strikte Diskriminierung zwischen Seryl- und Selenocysteyl-tRNASec durch SelB ist essentiell für das Funktionieren des Selenocystein inkorporierenden Systems. Eine gerichtete Mutatagenese der Aminoacyl-Bindetasche von SelB zeigte, dass die Selektivität der tRNA-Bindung vermutlich nicht auf einer spezifischen Erkennung des Aminoacyl-Rests beruht. Nach Zufallsmutagenese konnten vier SelB-Varianten isoliert werden, die in vivo eine erhöhte Aktivität mit Seryl-tRNASec besitzen. Zwei der Mutationen waren in der G-Domäne von SelB lokalisiert, die anderen beiden in Domäne 4a. Die biochemische Charakterisierung der mutierten Proteine ergab noch keinen Hinweis auf eine erhöhte Affinität der SelB-Varianten für Seryl-tRNASec, so dass andere Mechanismen für die Erweiterung der Aminosäure-Spezifität verantwortlich sein müssen. 2) Die Interaktion von SelB mit seinen Liganden wurde mit Hilfe von biochemischen und biophysikalischen Methoden analysiert. Der Elongationsfaktor zeigt im Gegensatz zu vielen anderen G-Proteinen eine höhere Affinität für GTP (KD = 0,74 µM) als für GDP (KD = 13,4 µM),was zusammen mit der hohen Dissoziationsrate von GDP (kdis = 15 s-1) darauf hinweist, dass der Nukleotidaustausch ohne Katalyse durch einen Austauschfaktor erfolgt. Die Kinetiken der Interaktion mit Guanosin-Nukleotiden werden durch die Gegenwart eines SECIS-Elements nicht beeinflusst. Die Affinität von SelB zu einem fluoresceinmarkierten SECIS-Transkript liegt im nanomolaren Bereich (KD = 1,23 nM), wobei die Assoziations- und Dissoziationskinetiken sehr schnell sind und durch die Gegenwart von Guanosin-Nukleotiden nicht verändert werden. In Gegenwart von Selenocysteyl-tRNASec wurde jedoch eine signifikante Verringerung der Dissoziationsgeschwindigkeit beobachtet, die zu einer Stabilisierung der Bindung führt und eine Interaktion zwischen der SECIS- und tRNA-Bindetasche nahelegt. Diese intramolekulare Wechselwirkung wurde durch Charakterisierung der isolierten mRNA-Bindedomäne von SelB bestätigt. Die Gleichgewichtslage der einzelnen Reaktionen führt zu einer gerichteten Bildung eines Komplexes aus SelB, GTP, Selenocysteyl-tRNASec und dem SECIS-Element, der durch seine hohe Stabilität auf der mRNA fixiert wird und gleichzeitig eine Konformation annimmt, die seine Interaktion mit dem Ribosom zulässt. 3) In der 5´-untranslatierten Region der selAB-mRNA wurde eine Sekundärstruktur identifiziert, die Ähnlichkeit mit dem SECIS-Element aufweist und mit der SelB spezifisch und mit hoher Affinität interagiert. Die Stabilität des Komplexes zwischen SelB und dem SECIS-ähnlichen Element erhöht sich in Gegenwart von Selenocysteyl-tRNASec. Eine Analyse der sel-Genexpression ergab, dass die Synthese von SelA und in geringerem Ausmaß SelB in genetischen Hintergründen, die eine Assemblierung des quaternären Komplexes aus SelB, GTP, Selenocysteyl- tRNASec und dem SECIS-ähnlichen Element erlauben, reprimiert ist. Mutationen in sel- Genen führen dagegen zu einer erhöhten intrazellulären Konzentration dieser Proteine. Mit Hilfe von Reportergen-Fusionen wurde gezeigt, dass die Repression der selA-Expression direkt von der Bildung eines quaternären Komplexes am SECIS-ähnlichen Element abhängig ist. Da diese keinen Einfluss auf die Transkription hat und nur zu einer schwachen Verringerung der mRNA-Menge führt, wurde gefolgert, dass das SECIS-ähnliche Element eine Regulation der Translationsinitiation am selA-Gen in Abhängigkeit vom Selenstatus der Zelle ermöglicht

Activated chemoreceptor arrays remain intact and hexagonally packed

Bacterial chemoreceptors cluster into exquisitively sensitive, tunable, highly ordered, polar arrays. While these arrays serve as paradigms of cell signalling in general, it remains unclear what conformational changes transduce signals from the periplasmic tips, where attractants and repellents bind, to the cytoplasmic signalling domains. Conflicting reports support and contest the hypothesis that activation causes large changes in the packing arrangement of the arrays, up to and including their complete disassembly. Using electron cryotomography, here we show that in Caulobacter crescentus, chemoreceptor arrays in cells grown in different media and immediately after exposure to the attractant galactose all exhibit the same 12 nm hexagonal packing arrangement, array size and other structural parameters. ΔcheB and ΔcheR mutants mimicking attractant- or repellent-bound states prior to adaptation also show the same lattice structure. We conclude that signal transduction and amplification must be accomplished through only small, nanoscale conformational changes

DipM, a new factor required for peptidoglycan remodelling during cell division in Caulobacter crescentus

In bacteria, cytokinesis is dependent on lytic enzymes that facilitate remodelling of the cell wall during constriction. In this work, we identify a thus far uncharacterized periplasmic protein, DipM, that is required for cell division and polarity in Caulobacter crescentus. DipM is composed of four peptidoglycan binding (LysM) domains and a C-terminal lysostaphin-like (LytM) peptidase domain. It binds to isolated murein sacculi in vitro, and is recruited to the site of constriction through interaction with the cell division protein FtsN. Mutational analyses showed that the LysM domains are necessary and sufficient for localization of DipM, while its peptidase domain is essential for function. Consistent with a role in cell wall hydrolysis, DipM was found to interact with purified murein sacculi in vitro and to induce cell lysis upon overproduction. Its inactivation causes severe defects in outer membrane invagination, resulting in a significant delay between cytoplasmic compartmentalization and final separation of the daughter cells. Overall, these findings indicate that DipM is a periplasmic component of the C. crescentus divisome that facilitates remodelling of the peptidoglycan layer and, thus, coordinated constriction of the cell envelope during the division process

General Protein Diffusion Barriers Create Compartments within Bacterial Cells

In eukaryotes, the differentiation of cellular extensions such as cilia or neuronal axons depends on the partitioning of proteins to distinct plasma membrane domains by specialized diffusion barriers. However, examples of this compartmentalization strategy are still missing for prokaryotes, although complex cellular architectures are also widespread among this group of organisms. This study reveals the existence of a protein-mediated membrane diffusion barrier in the stalked bacterium Caulobacter crescentus. We show that the Caulobacter cell envelope is compartmentalized by macromolecular complexes that prevent the exchange of both membrane and soluble proteins between the polar stalk extension and the cell body. The barrier structures span the cross-sectional area of the stalk and comprise at least four proteins that assemble in a cell-cycle-dependent manner. Their presence is critical for cellular fitness because they minimize the effective cell volume, allowing faster adaptation to environmental changes that require de novo synthesis of envelope proteins

Dynamic metabolic rewiring enables efficient acetyl-CoA assimilation in Paracoccus denitrificans

During growth, microorganisms have to balance metabolic flux between energy and biosynthesis. One of the key intermediates in central carbon metabolism is acetyl-CoA, which can be either oxidized in the citric acid cycle or assimilated into biomass through dedicated pathways. Two acetyl-CoA assimilation strategies have been described in bacteria so far, the ethylmalonyl-CoA pathway (EMCP) and the glyoxylate cycle (GC). Here, we show that Paracoccus denitrificans uses both strategies for acetyl-CoA assimilation during different growth stages, revealing an unexpected metabolic complexity in the organism’s central carbon metabolism. The EMCP is constitutively expressed on various substrates and leads to high biomass yields on substrates requiring acetyl-CoA assimilation, such as acetate, while the GC is specifically induced on these substrates, enabling fast growth rates. Even though each acetyl-CoA assimilation strategy alone confers a distinct growth advantage, P. denitrificans recruits both to adapt to changing environmental conditions, such as a switch from succinate to acetate. Time-resolved single-cell experiments show that during this switch, expression of the EMCP and GC is highly coordinated, indicating fine-tuned genetic programming. The dynamic metabolic rewiring of acetyl-CoA assimilation is an evolutionary innovation by P. denitrificans that allows this organism to respond in a highly flexible manner to changes in the nature and availability of the carbon source to meet the physiological needs of the cell, representing a new phenomenon in central carbon metabolism

SMC is recruited to oriC by ParB and promotes chromosome segregation in Streptococcus pneumoniae

Segregation of replicated chromosomes is an essential process in all organisms. How bacteria, such as the oval-shaped human pathogen Streptococcus pneumoniae, efficiently segregate their chromosomes is poorly understood. Here we show that the pneumococcal homologue of the DNA-binding protein ParB recruits S. pneumoniae condensin (SMC) to centromere-like DNA sequences (parS) that are located near the origin of replication, in a similar fashion as was shown for the rod-shaped model bacterium Bacillus subtilis. In contrast to B. subtilis, smc is not essential in S. pneumoniae, and Δsmc cells do not show an increased sensitivity to gyrase inhibitors or high temperatures. However, deletion of smc and/or parB results in a mild chromosome segregation defect. Our results show that S. pneumoniae contains a functional chromosome segregation machine that promotes efficient chromosome segregation by recruitment of SMC via ParB. Intriguingly, the data indicate that other, as of yet unknown mechanisms, are at play to ensure proper chromosome segregation in this organism.

Spatial and topological organization of DNA chains induced by gene co-localization

Transcriptional activity has been shown to relate to the organization of chromosomes in the eukaryotic nucleus and in the bacterial nucleoid. In particular, highly transcribed genes, RNA polymerases and transcription factors gather into discrete spatial foci called transcription factories. However, the mechanisms underlying the formation of these foci and the resulting topological order of the chromosome remain to be elucidated. Here we consider a thermodynamic framework based on a worm-like chain model of chromosomes where sparse designated sites along the DNA are able to interact whenever they are spatially close-by. This is motivated by recurrent evidence that there exists physical interactions between genes that operate together. Three important results come out of this simple framework. First, the resulting formation of transcription foci can be viewed as a micro-phase separation of the interacting sites from the rest of the DNA. In this respect, a thermodynamic analysis suggests transcription factors to be appropriate candidates for mediating the physical interactions between genes. Next, numerical simulations of the polymer reveal a rich variety of phases that are associated with different topological orderings, each providing a way to increase the local concentrations of the interacting sites. Finally, the numerical results show that both one-dimensional clustering and periodic location of the binding sites along the DNA, which have been observed in several organisms, make the spatial co-localization of multiple families of genes particularly efficient.Comment: Figures and Supplementary Material freely available on http://dx.doi.org/10.1371/journal.pcbi.100067

Dynamics of the peptidoglycan biosynthetic machinery in the stalked budding bacterium Hyphomonas neptunium

Most commonly studied bacteria grow symmetrically and divide by binary fission, generating two siblings of equal morphology. An exception to this rule are budding bacteria, in which new offspring emerges de novo from a morphologically invariant mother cell. Although this mode of proliferation is widespread in diverse bacterial lineages, the underlying mechanisms are still incompletely understood. Here, we perform the first molecular-level analysis of growth and morphogenesis in the stalked budding alphaproteobacterium Hyphomonas neptunium. Peptidoglycan labeling shows that, in this species, buds originate from a stalk-like extension of the mother cell whose terminal segment is gradually remodeled into a new cell compartment. As a first step toward identifying the machinery mediating the budding process, we performed comprehensive mutational and localization studies of predicted peptidoglycan biosynthetic proteins in H. neptunium. These analyses identify factors that localize to distinct zones of dispersed and zonal growth, and they suggest a critical role of the MreB-controlled elongasome in cell morphogenesis. Collectively, our work shows that the mechanism of growth in H. neptunium is distinct from that in related, polarly growing members of the order Rhizobiales, setting the stage for in-depth analyses of the molecular principles regulating the fascinating developmental cycle of this species