Metabolic fluxes in the central carbon metabolism of Dinoroseobacter shibae and Phaeobacter gallaeciensis, two members of the marine Roseobacter clade

<p>Abstract</p> <p>Background</p> <p>In the present work the central carbon metabolism of <it>Dinoroseobacter shibae </it>and <it>Phaeobacter gallaeciensis </it>was studied at the level of metabolic fluxes. These two strains belong to the marine <it>Roseobacter </it>clade, a dominant bacterial group in various marine habitats, and represent surface-associated, biofilm-forming growth (<it>P. gallaeciensis</it>) and symbiotic growth with eukaryotic algae (<it>D. shibae</it>). Based on information from recently sequenced genomes, a rich repertoire of pathways has been identified in the carbon core metabolism of these organisms, but little is known about the actual contribution of the various reactions <it>in vivo</it>.</p> <p>Results</p> <p>Using ¹³C labelling techniques in specifically designed experiments, it could be shown that glucose-grown cells of <it>D. shibae </it>catabolise the carbon source exclusively via the Entner-Doudoroff pathway, whereas alternative routes of glycolysis and the pentose phosphate pathway are obviously utilised for anabolic purposes only. Enzyme assays confirmed this flux pattern and link the lack of glycolytic flux to the absence of phosphofructokinase activity. The previously suggested formation of phosphoenolpyruvate from pyruvate during mixotrophic CO₂assimilation was found to be inactive under the conditions studied. Moreover, it could be shown that pyruvate carboxylase is involved in CO₂assimilation and that the <it>cyclic </it>respiratory mode of the TCA cycle is utilised. Interestingly, the use of intracellular pathways was highly similar for <it>P. gallaeciensis</it>.</p> <p>Conclusion</p> <p>The present study reveals the first insight into pathway utilisation within the <it>Roseobacter </it>group. Fluxes through major intracellular pathways of the central carbon metabolism, which are closely linked to the various important traits found for the <it>Roseobacter </it>clade, could be determined. The close similarity of fluxes between the two physiologically rather different species might provide the first indication of more general key properties among members of the <it>Roseobacter </it>clade which may explain their enormous success in the marine realm.</p

Integrated Transcriptional Regulatory Network of Quorum Sensing, Replication Control, and SOS Response in Dinoroseobacter shibae

Quorum sensing (QS) coordinates population wide gene expression of bacterial species. Highly adaptive traits like gene transfer agents (GTA), morphological heterogeneity, type 4 secretion systems (T4SS), and flagella are QS controlled in Dinoroseobacter shibae, a Roseobacter model organism. Its QS regulatory network is integrated with the CtrA phosphorelay that controls cell division in alphaproteobacteria. To elucidate the network topology, we analyzed the transcriptional response of the QS-negative D. shibae strain ΔluxI1 toward externally added autoinducer (AI) over a time period of 3 h. The signaling cascade is initiated by the CtrA phosphorelay, followed by the QS genes and other target genes, including the second messenger c-di-GMP, competence, flagella and pili. Identification of transcription factor binding sites in promoters of QS induced genes revealed the integration of QS, CtrA phosphorelay and the SOS stress response mediated by LexA. The concentration of regulatory genes located close to the origin or terminus of replication suggests that gene regulation and replication are tightly coupled. Indeed, addition of AI first stimulates and then represses replication. The restart of replication comes along with increased c-di-GMP levels. We propose a model in which QS induces replication followed by differentiation into GTA producing and non-producing cells. CtrA-activity is controlled by the c-di-GMP level, allowing some of the daughter cells to replicate again. The size of the GTA producing subpopulation is tightly controlled by QS via the AI Synthase LuxI2. Finally, induction of the SOS response allows for integration of GTA DNA into the host chromosome

Genomic epidemiology of clinical ESBL-producing Enterobacteriaceae in a German hospital suggests infections are primarily community- and regionally-acquired

Clinical Enterobacteriaceae isolates that produce extended-spectrum β-lactamases (ESBLs) have been increasingly reported at a global scale. However, comprehensive data on the molecular epidemiology of ESBL-producing strains are limited and few studies have been conducted in non-outbreak situations. We used whole-genome sequencing to describe the population structure of 294 ESBL-producing Escherichia coli and Klebsiella pneumoniae isolates that were recovered from a German community hospital throughout a 1 year sampling period in a non-outbreak situation. We found a high proportion of E. coli isolates (61.5 %) belonged to the globally disseminated extraintestinal pathogenic ST131, whereas a wider diversity of STs was observed among K. pneumoniae isolates. The E. coli ST131 population in this study was shaped by multiple introductions of strains as demonstrated by contextual genomic analysis including ST131 strains from other geographical sources. While no recent common ancestor of the isolates of the current study and other international isolates was found, our clinical isolates clustered with those previously recovered in the region. Furthermore, we found that the isolation of ESBL-producing clinical strains in hospitalized patients could only rarely be associated with likely patient-to-patient transmission, indicating primarily a community and regional acquisition of strains. Further genomic analyses of clinical, carriage and environmental isolates is needed to uncover hidden transmissions and thus discover the most common sources of ESBL-producing pathogen infections in our hospitals

Strategi Pembangunan Pariwisata melalui Sinergitas Dinas Pariwisata dengan Desa Adat ( Studi Kasus pada Pengelolaan Obyek Wisata Pantai Labuan Sait dalam Meningkatkan Retribusi Daerah di Kabupaten Badung)

A gradual Tourism development is very important to improve the quality of tourism each year to compete with other tourist attraction. The synergy between the Central Government with local government plays an important role to the development of tourism. The background to this research is the development of tourism which is still insufficient in Labuan Sait both in terms of means and infrastructure, promotion, as well as structuring tourism. This study measures how does tourism development strategy through the synergy with the customary village tourism office on the management of Beach Tourism Labuan Sait in increasing the levy County in Badung Regency with the theory of development that uses the concept of planning development by Sjahrizal in the regional development planning in the era of autonomy. The indicator consists of planning, implementation, monitoring and evaluation. In addition also use the concept of synergy from Najiyati and Rahmat which consists of indicators communication and coordination as well as indicators of the SWOT by Freddy Rangkuti. Method used in this study is a qualitative method with descriptive approach with data collection techniques in the form of in-depth interviews to several informants associated with this research. The results of the research showed that the development strategy of tourism through the synergy with the customary village Tourism Office on the management of Beach Tourism Labuan Sait in improving regional levies in Badung Regency are still insufficient. That is because the is still lacking from the indicator monitoring and implementation and evaluation of the impact against the decline of levy of admission attractions Labuan Sait in the 2017. &nbsp; &nbsp; Keywords: Development, Tourism, Synergy, and Strateg

Organism-specific depletion of highly abundant RNA species from bacterial total RNA.

High-throughput sequencing has become a standard tool for transcriptome analysis. The depletion of overrepresented RNA species from sequencing libraries plays a key role in establishing potent and cost-efficient RNA-seq routines. Commercially available kits are known to obtain good results for the reduction of ribosomal RNA (rRNA). However, we found that the transfer-messenger RNA (tmRNA) was frequently highly abundant in rRNA-depleted samples of Pseudomonas aeruginosa , consuming up to 25 % of the obtained reads. The tmRNA fraction was particularly high in samples taken from stationary cultures. This suggests that overrepresentation of this RNA species reduces the mRNA fraction when cells are grown under challenging conditions. Here, we present an RNase-H-based depletion protocol that targets the tmRNA in addition to ribosomal RNAs. We were able to increase the mRNA fraction to 93-99% and therefore outperform not only the commercially Ribo-off kit (Vazyme) operating by the same principle but also the formerly widely used Ribo-Zero kit (Illumina). Maximizing the read share of scientifically interesting RNA species enhances the discriminatory potential of next-generation RNA-seq experiments and, therefore, can contribute to a better understanding of the transcriptomic landscape of bacterial pathogens and their used mechanisms in host infection

Connection Between Chromosomal Location and Function of CtrA Phosphorelay Genes in Alphaproteobacteria.

Most bacterial chromosomes are circular, with replication starting at one origin (ori) and proceeding on both replichores toward the terminus (ter). Several studies have shown that the location of genes relative to ori and ter can have profound effects on regulatory networks and physiological processes. The CtrA phosphorelay is a gene regulatory system conserved in most alphaproteobacteria. It was first discovered in Caulobacter crescentus where it controls replication and division into a stalked and a motile cell in coordination with other factors. The locations of the ctrA gene and targets of this response regulator on the chromosome affect their expression through replication-induced DNA hemi-methylation and specific positioning along a CtrA activity gradient in the dividing cell, respectively. Here we asked to what extent the location of CtrA regulatory network genes might be conserved in the alphaproteobacteria. We determined the locations of the CtrA phosphorelay and associated genes in closed genomes with unambiguously identifiable ori from members of five alphaproteobacterial orders. The location of the phosphorelay genes was the least conserved in the Rhodospirillales followed by the Sphingomonadales. In the Rhizobiales a trend toward certain chromosomal positions could be observed. Compared to the other orders, the CtrA phosphorelay genes were conserved closer to ori in the Caulobacterales. In contrast, the genes were highly conserved closer to ter in the Rhodobacterales. Our data suggest selection pressure results in differential positioning of CtrA phosphorelay and associated genes in alphaproteobacteria, particularly in the orders Rhodobacterales, Caulobacterales and Rhizobiales that is worth deeper investigation

The Alternative Sigma Factor SigX Controls Bacteriocin Synthesis and Competence, the Two Quorum Sensing Regulated Traits in Streptococcus mutans.

Two small quorum sensing (QS) peptides regulate competence in S. mutans in a cell density dependent manner: XIP (sigX inducing peptide) and CSP (competence stimulating peptide). Depending on the environmental conditions isogenic S. mutans cells can split into a competent and non-competent subpopulation. The origin of this population heterogeneity has not been experimentally determined and it is unknown how the two QS systems are connected. We developed a toolbox of single and dual fluorescent reporter strains and systematically knocked out key genes of the competence signaling cascade in the reporter strain backgrounds. By following signal propagation on the single cell level we discovered that the master regulator of competence, the alternative sigma factor SigX, directly controls expression of the response regulator for bacteriocin synthesis ComE. Consequently, a SigX binding motif (cin-box) was identified in the promoter region of comE. Overexpressing the genetic components involved in competence development demonstrated that ComRS represents the origin of bimodality and determines the modality of the downstream regulators SigX and ComE. Moreover these analysis showed that there is no direct regulatory link between the two QS signaling cascades. Competence is induced through a hierarchical XIP signaling cascade, which has no regulatory input from the CSP cascade. CSP exclusively regulates bacteriocin synthesis. We suggest renaming it mutacin inducing peptide (MIP). Finally, using phosphomimetic comE mutants we show that unimodal bacteriocin production is controlled posttranslationally, thus solving the puzzling observation that in complex media competence is observed in a subpopulation only, while at the same time all cells produce bacteriocins. The control of both bacteriocin synthesis and competence through the alternative sigma-factor SigX suggests that S. mutans increases its genetic repertoire via QS controlled predation on neighboring species in its natural habitat