Simple generation of site-directed point mutations in the Escherichia coli chromosome using Red®/ET® Recombination

Background: Introducing point mutations into bacterial chromosomes is important for further progress in studies relying on functional genomics, systems- and synthetic biology, and for metabolic engineering. For many investigations, chromosomal systems are required rather than artificial plasmid based systems. Results: Here we describe the introduction of a single point mutation into the Escherichia coli chromosome by site-directed mutagenesis without leaving any selection marker. We used Red (R)/ET (R) Recombination in combination with rpsL counter-selection to introduce a single point mutation into the E. coli MG1655 genome, one of the widely used bacterial model strains in systems biology. The method we present is rapid and highly efficient. Since single-stranded synthetic oligonucleotides can be used for recombination, any chromosomal modification can be designed. Conclusion: Chromosomal modifications performed by rpsL counter-selection may also be used for other bacteria that contain an rpsL homologue, since Red (R)/ET (R) Recombination has been applied to several enteric bacteria before

Single cell analysis of Vibrio harveyi uncovers functional heterogeneity in response to quorum sensing signals

Background: Vibrio harveyi and closely related species are important pathogens in aquaculture. A complex quorum sensing cascade involving three autoinducers controls bioluminescence and several genes encoding virulence factors. Single cell analysis of a V. harveyi population has already indicated intercellular heterogeneity in the production of bioluminescence. This study was undertaken to analyze the expression of various autoinducer-dependent genes in individual cells. Results: Here we used reporter strains bearing promoter::gfp fusions to monitor the induction/repression of three autoinducer-regulated genes in wild type conjugates at the single cell level. Two genes involved in pathogenesis - vhp and vscP, which code for an exoprotease and a component of the type III secretion system, respectively, and luxC (the first gene in the lux operon) were chosen for analysis. The lux operon and the exoprotease gene are induced, while vscP is repressed at high cell density. As controls luxS and recA, whose expression is not dependent on autoinducers, were examined. The responses of the promoter:: gfp fusions in individual cells from the same culture ranged from no to high induction. Importantly, simultaneous analysis of two autoinducer induced phenotypes, bioluminescence (light detection) and exoproteolytic activity (fluorescence of a promoter:: gfp fusion), in single cells provided evidence for functional heterogeneity within a V. harveyi population. Conclusions: Autoinducers are not only an indicator for cell density, but play a pivotal role in the coordination of physiological activities within the population

Domain swapping reveals that the N-terminal domain of the sensor kinase KdpD in Escherichia coli is important for signaling

<p>Abstract</p> <p>Background</p> <p>The KdpD/KdpE two-component system of <it>Escherichia coli </it>regulates expression of the <it>kdpFABC </it>operon encoding the high affinity K⁺transport system KdpFABC. The input domain of KdpD comprises a domain that belongs to the family of universal stress proteins (Usp). It has been previously demonstrated that UspC binds to this domain, resulting in KdpD/KdpE scaffolding under salt stress. However the mechanistic significance of this domain for signaling remains unclear. Here, we employed a "domain swapping" approach to replace the KdpD-Usp domain with four homologous domains or with the six soluble Usp proteins of <it>E. coli</it>.</p> <p>Results</p> <p>Full response to salt stress was only achieved with a chimera that contains UspC, probably due to unaffected scaffolding of the KdpD/KdpE signaling cascade by soluble UspC. Unexpectedly, chimeras containing either UspF or UspG not only prevented <it>kdpFABC </it>expression under salt stress but also under K⁺limiting conditions, although these hybrid proteins exhibited kinase and phosphotransferase activities <it>in vitro</it>. These are the first KdpD derivatives that do not respond to K⁺limitation due to alterations in the N-terminal domain. Analysis of the KdpD-Usp tertiary structure revealed that this domain has a net positively charged surface, while UspF and UspG are characterized by net negative surface charges.</p> <p>Conclusion</p> <p>The Usp domain within KdpD not only functions as a binding surface for the scaffold UspC, but it is also important for KdpD signaling. We propose that KdpD sensing/signaling involves alterations of electrostatic interactions between the large N- and C-terminal cytoplasmic domains.</p

Detection and function of an intramolecular disulfide bond in the pH-responsive CadC of Escherichia coli

Background: In an acidic and lysine-rich environment Escherichia coli induces expression of the cadBA operon which encodes CadA, the lysine decarboxylase, and CadB, the lysine/cadaverine antiporter. cadBA expression is dependent on CadC, a membrane-integrated transcriptional activator which belongs to the ToxR-like protein family. Activation of CadC requires two stimuli, lysine and low pH. Whereas lysine is detected by an interplay between CadC and the lysine-specific transporter LysP, pH alterations are sensed by CadC directly. Crystal structural analyses revealed a close proximity between two periplasmic cysteines, Cys208 and Cys272. Results: Substitution of Cys208 and/or Cys272 by alanine resulted in CadC derivatives that were active in response to only one stimulus, either lysine or pH 5.8. Differential in vivo thiol trapping revealed a disulfide bond between these two residues at pH 7.6, but not at pH 5.8. When Cys208 and Cys272 were replaced by aspartate and lysine, respectively, virtually wild-type behavior was restored indicating that the disulfide bond could be mimicked by a salt bridge. Conclusion: A disulfide bond was found in the periplasmic domain of CadC that supports an inactive state of CadC at pH 7.6. At pH 5.8 disulfide bond formation is prevented which transforms CadC into a semi-active state. These results provide new insights into the function of a pH sensor

Opportunities and Challenges to Profile mRNA Modifications in Escherichia coli

mRNA methylation is an important regulator of many physiological processes in eukaryotes but has not been studied in depth in prokaryotes. Working with bacterial mRNA is challenging because it lacks a poly(A)-tail. However, methods for detecting RNA modifications, both sequencing and mass spectrometry, rely on efficient preparation of mRNA. Here, we compared size-dependent separation by electrophoresis and rRNA depletion for enrichment of Escherichia coli mRNA. The purification success was monitored by qRT-PCR and RNA sequencing. Neither method allowed complete removal of rRNA. Nevertheless, we were able to quantitatively analyze several modified nucleosides in the different RNA types. We found evidence for stress dependent RNA modification reprofiling in rRNA, but also several modified nucleosides in the mRNA enriched fractions showed significant changes

Structurally rich dry grasslands – Potential stepping stones for bats in open farmland

Agricultural intensification has caused decrease and fragmentation of European semi-natural dry grasslands. While a high biodiversity value of dry grasslands is acknowledged for plants and insects, locally and on landscape level, their relevance for mobile species, such as bats, is unknown. Here we investigate the use of dry grassland fragments by bats in an agriculturally intensified region in Germany and evaluate local and landscape factors influencing bat activity and assemblages. Specifically, we predicted that a combination of local dry grassland structural richness and landscape features as well as their interactions affect bat activity and foraging above dry grasslands. We also expected that these features influence compositions of local bat assemblages. We repeatedly sampled at 12 dry grassland plots with acoustic monitoring and assessed activity and foraging of bat species/sonotypes, which we grouped into guilds known for foraging in open land, at vegetation edges and in narrow spaces. We determined structural richness of the dry grassland plots in field and derived landscape features from digital landscape data. A relatively high proportion of bat species/sonotypes used dry grasslands regularly. The edge space foragers responded positively to higher local structural richness. Their dry grassland use increased when surrounding forests and woody features were less available, but they foraged more on dry grasslands closer to water bodies. Narrow space bat activity on dry grasslands decreased with less landscape connectivity. Open and narrow space foragers responded to local structural richness only in landscape context. For all bat guilds we found increased use of structurally richer dry grasslands when there was more open farmland in the surroundings. This was also the case for edge space foragers, when landscapes were more homogeneous. Lastly, with increasing structural richness, bat assemblages were more dominated by edge space foragers. We show the importance of European dry grassland fragments for the highly mobile group of bats under certain local structural and landscape compositional conditions. Our results underline the value of heterogeneous dry grassland fragments as potential stepping stones in intensively used farmland areas and contribute to evidence based decision making in dry grassland management and bat conservation

Photorhabdus luminescens genes induced upon insect infection

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Urban areas in rural landscapes – the importance of green space and local architecture for bat conservation

Urbanization is a highly disperse process, resulting in urban sprawl across landscapes. Within such landscapes, structural heterogeneity may be an important factor for maintaining biodiversity. We investigated the importance of habitat heterogeneity on bats in villages across the Schwäbische Alb, Germany, a progressively urbanized region. Bat activity and diversity were assessed using acoustic monitoring. We characterized habitat composition at the local and neighborhood scale and assessed environmental characteristics of urban density, vegetation cover and architectural features, combining satellite and ground-based measures. Our results revealed that the extent of urban areas determines the occurrence of different bat species, while local spatial, structural, and architectonic parameters at recording sites affected bat activity, feeding activity and social encounters. Larger urban areas with increased proportion of impervious surfaces and newly constructed housing areas were associated with fewer bat species and lower bat activity. Bat activity and feeding were highest in housing areas constructed between 1950-2000 and increased with higher proportions of older, rather openly structured vegetation. Our results clearly show a combined importance of environmental parameters across spatial scales, affecting habitat suitability and quality of rural urban areas for bats. This highlights that strategies for biodiversity inclusion in rural urban planning need to consider both local and neighborhood conditions to support bat diversity and vital bat activity. In particular, it exemplifies future challenges to maintain biodiversity within progressively urbanized rural landscapes, as this needs support by municipalities for maintaining space for nature in areas designated for urban development and also the consciousness by local residents for biodiversity-friendly modernizations

The role of polyproline motifs in the histidine kinase EnvZ

Although distinct amino acid motifs containing consecutive prolines (polyP) cause ribosome stalling, which necessitates recruitment of the translation elongation factor P (EF-P), they occur strikingly often in bacterial proteomes. For example, polyP motifs are found in more than half of all histidine kinases in Escherichia coli K-12, which raises the question of their role(s) in receptor function. Here we have investigated the roles of two polyP motifs in the osmosensor and histidine kinase EnvZ. We show that the I PPPL motif in the HAMP domain is required for dimerization of EnvZ. Moreover, replacement of the prolines in this motif by alanines disables the receptor's sensor function. The second motif, VVPPA, which is located in the periplasmic domain, was found to be required for interaction with the modulator protein MzrA. Our study also reveals that polyP-dependent stalling has little effect on EnvZ levels. Hence, both polyP motifs in EnvZ are primarily involved in protein-protein interaction. Furthermore, while the first motif occurs in almost all EnvZ homologues, the second motif is only found in species that have MzrA, indicating co-evolution of the two proteins

NAIL-MS reveals the repair of 2-methylthiocytidine by AlkB in E. coli

RNAs contain post-transcriptional modifications, which fulfill a variety of functions in translation, secondary structure stabilization and cellular stress survival. Here, 2-methylthiocytidine (ms(2)C) is identified in tRNA of E. coli and P. aeruginosa using NAIL-MS (nucleic acid isotope labeling coupled mass spectrometry) in combination with genetic screening experiments. ms(2)C is only found in 2-thiocytidine (s(2)C) containing tRNAs, namely tRNA(CCG)(Arg), tRNA(ICG)(Ar)(g), tRNA(UCU)(Arg) and tRNA(GCU)(Ser )at low abundances. ms(2)C is not formed by, commonly known tRNA methyltransferases. Instead, we observe its formation in vitro and in vivo during exposure to methylating agents. More than half of the s(2)C containing tRNA can be methylated to carry ms(2)C. With a pulse-chase NAIL-MS experiment, the repair mechanism by AlkB dependent sulfur demethylation is demonstrated in vivo. Overall, we describe ms(2)C as a bacterial tRNA modification and damage product. Its repair by AlkB and other pathways is demonstrated in vivo by our powerful NAIL-MS approach