Photorhabdus luminescens is an enteric bacterium, which lives in mutualistic association with soil nematodes and is highly pathogenic for a broad spectrum of insects. A complete genome sequence for ...the type strain P. luminescens subsp. laumondii TT01, which was originally isolated in Trinidad and Tobago, has been described earlier. Subsequently, a rifampicin resistant P. luminescens strain has been generated with superior possibilities for experimental characterization. This strain, which is widely used in research, was described as a spontaneous rifampicin resistant mutant of TT01 and is known as TT01-Rif
.
Unexpectedly, upon phenotypic comparison between the rifampicin resistant strain and its presumed parent TT01, major differences were found with respect to bioluminescence, pigmentation, biofilm formation, haemolysis as well as growth. Therefore, we renamed the strain TT01-Rif
to DJC. To unravel the genomic basis of the observed differences, we generated a complete genome sequence for strain DJC using the PacBio long read technology. As strain DJC was supposed to be a spontaneous mutant, only few sequence differences were expected. In order to distinguish these from potential sequencing errors in the published TT01 genome, we re-sequenced a derivative of strain TT01 in parallel, also using the PacBio technology. The two TT01 genomes differed at only 30 positions. In contrast, the genome of strain DJC varied extensively from TT01, showing 13,000 point mutations, 330 frameshifts, and 220 strain-specific regions with a total length of more than 300 kb in each of the compared genomes.
According to the major phenotypic and genotypic differences, the rifampicin resistant P. luminescens strain, now named strain DJC, has to be considered as an independent isolate rather than a derivative of strain TT01. Strains TT01 and DJC both belong to P. luminescens subsp. laumondii.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Purpose
Phenotypic heterogeneity occurs in many bacterial populations: single cells of the same species display different phenotypes, despite being genetically identical. The Gram-negative ...entomopathogenic bacterium
Photorhabdus luminescens
is an excellent example to investigate bacterial phenotypic heterogeneity. Its dualistic life cycle includes a symbiotic stage interacting with entomopathogenic nematodes (EPNs) and a pathogenic stage killing insect larvae.
P. luminescens
appears in two phenotypically different cell forms: the primary (1°) and the secondary (2°) cell variants. While 1° cells are bioluminescent, pigmented, and produce a huge set of secondary metabolites, 2° cells lack all these phenotypes. The main difference between both phenotypic variants is that only 1° cells can undergo symbiosis with EPNs, a phenotype that is absent from 2° cells. Recent comparative transcriptome analysis revealed that genes mediating 1° cell-specific traits are modulated differently in 2° cells. Although it was previously suggested that heterogeneity in
P. luminescens
cells cultures is not genetically mediated by, e.g., larger rearrangements in the genome, the genetic similarity of both cell variants has not clearly been demonstrated yet.
Methods
Here, we analyzed the genomes of both 1° and 2° cells by genome sequencing of each six single 1° and 2° clones that emerged from a single 1° clone after prolonged growth. Using different bioinformatics tools, the sequence data were analyzed for clustered point mutations or genetic rearrangements with respect to the respective phenotypic variant.
Result
We demonstrate that isolated clones of 2° cells that switched from the 1° cell state do not display any noticeable mutation and do not genetically differ from 1° cells.
Conclusion
In summary, we show that the phenotypic differences in
P. luminescens
cell cultures are obviously not caused by mutations or genetic rearrangements in the genome but truly emerge from phenotypic heterogeneity.
Phenotypic heterogeneity in microbial communities enables genetically identical organisms to behave differently even under the same environmental conditions. Photorhabdus luminescens, a ...bioluminescent Gram-negative bacterium, contains a complex life cycle, which involves a symbiotic interaction with nematodes as well as a pathogenic association with insect larvae. P. luminescens exists in two distinct phenotypic cell types, designated as the primary (1°) and secondary (2°) cells. The 1° cells are bioluminescent, pigmented and can support nematode growth and development. Individual 1° cells undergo phenotypic switching after prolonged cultivation and convert to 2° cells, which lack the 1° specific phenotypes. The LysR-type regulator HexA has been described as major regulator of this switching process. Here we show that HexA controls phenotypic heterogeneity in a versatile way, directly and indirectly. Expression of hexA is enhanced in 2° cells, and the corresponding regulator inhibits 1° specific traits in 2° cells. HexA does not directly affect bioluminescence, a predominant 1° specific phenotype. Since the respective luxCDABE operon is repressed at the post-transcriptional level and transcriptional levels of the RNA chaperone gene hfq are also enhanced in 2° cells, small regulatory RNAs are presumably involved that are under control of HexA. Another phenotypic trait that is specific for 1° cells is quorum sensing mediated cell clumping. The corresponding pcfABCDEF operon could be identified as the first direct target of HexA, since the regulator binds to the pcfA promoter region and thereby blocks expression of the target operon. In summary, our data show that HexA fulfills the task as repressor of 1° specific features in 2° cells in a versatile way and gives first insights into the complexity of regulating phenotypic heterogeneity in Photorhabdus bacteria.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Cross-sectional and longitudinal profiling of full sets of nucleic acids, peptides, or proteins as well as metabolites expressed in biospecimens acquired via a cardiovascular disease-oriented biobank ...may aid in the elucidation of the disease pathways and mechanisms underlying individual cardiovascular diseases, such as degenerative valvular heart disease. This may promote the development of novel and effective, personalized diagnostic and therapeutic strategies to efficiently reduce cardiovascular mortality and morbidity as well as its health and economic burden. This brief report aims to describe the unique, standardized, interdisciplinary, and interprofessional approach to cross-sectional and longitudinal cardiovascular biobanking and databasing at the University Hospital Augsburg. Moreover, we present the study protocol of a specific, well-defined, prospective, single-center research project involving cross-sectional and longitudinal cardiovascular biobanking. The aim of this project is to gain a better insight into the molecular mechanisms underlying aortic valve disease-induced cardiomyopathy and the long-term effect of surgical correction of the aortic valve pathology on the left ventricular myocardial molecule profile.
Abstract Objectives Liquid biopsy (LBx) provides diagnostic, prognostic and predictive insights for malignant diseases and offers promising applications regarding tumor burden, tumor heterogeneity ...and clonal evolution. Methods The Augsburg Longitudinal Plasma Study (ALPS) is a prospective trial for patients with metastatic cancer that comprises sequential collection of LBx samples, tumor tissue, radiological imaging data, clinical information and patient-reported outcomes. Peripheral blood plasma is collected based on the individual patient’s staging intervals and LBx-derived ctDNA analyses are performed using CAncer Personalized Profiling sequencing (CAPP-seq). Results From April 2021 to October 2023, 419 patients have been enrolled. A total of 1,293 LBx samples were collected, 419 samples (100 %) at the beginning of the study and an average of 3 (range 1–12) during the 30-month follow-up period of the current interim analysis. 380 tissue biopsy (TBx) samples (90.7 %) were available at baseline and 39.6 % had ≥1 TBx samples at follow-up. Lung cancer patients are most prevalent in ALPS (n=147), followed by colorectal (n=38), prostate (n=31) and gastroesophageal cancer (n=28). On average, 12.0 ng/mL plasma cell-free DNA (cfDNA) could be isolated. First CAPP-seq analyses in 60 patients comprised 110 samples and demonstrated a detection sensitivity of 0.1 %. Conclusions The first interim analysis of ALPS confirms feasibility for comprehensive longitudinal evaluation of LBx and demonstrates suitability for ctDNA evaluation.
The diagnosis of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), originally based on morphological assessment alone, has to bring together more and more disciplines. Today, modern ...AML/MDS diagnostics rely on cytomorphology, cytochemistry, immunophenotyping, cytogenetics, and molecular genetics. Only the integration of all these methods allows a comprehensive and complementary characterization of each case, which is a prerequisite for optimal AML/MDS diagnosis and treatment. In the following, we present why multidisciplinary and local diagnosis is essential today and will become even more important in the future, especially in the context of precision medicine. We present our idea and strategy implemented at Augsburg University Hospital, which has realized multidisciplinary diagnostics in AML/MDS in an interdisciplinary and decentralized approach. In particular, this includes the recent technical advances that molecular genetics provides with modern methods. The enormous amount of data generated by these techniques represents a major challenge, but also a unique opportunity. We will reflect on how this increase in knowledge can be integrated into routine practice to lead the way for personalized medicine in AML/MDS to improve patient care.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Background: Photorhabdus luminescens is an enteric bacterium, which lives in mutualistic association with soil nematodes and is highly pathogenic for a broad spectrum of insects. A complete genome ...sequence for the type strain P. luminescens subsp. laumondii TT01, which was originally isolated in Trinidad and Tobago, has been described earlier. Subsequently, a rifampicin resistant P. luminescens strain has been generated with superior possibilities for experimental characterization. This strain, which is widely used in research, was described as a spontaneous rifampicin resistant mutant of TT01 and is known as TT01-Rif(R).Results: Unexpectedly, upon phenotypic comparison between the rifampicin resistant strain and its presumed parent TT01, major differences were found with respect to bioluminescence, pigmentation, biofilm formation, haemolysis as well as growth. Therefore, we renamed the strain TT01-Rif(R) to DJC. To unravel the genomic basis of the observed differences, we generated a complete genome sequence for strain DJC using the PacBio long read technology. As strain DJC was supposed to be a spontaneous mutant, only few sequence differences were expected. In order to distinguish these from potential sequencing errors in the published TT01 genome, we re-sequenced a derivative of strain TT01 in parallel, also using the PacBio technology. The two TT01 genomes differed at only 30 positions. In contrast, the genome of strain DJC varied extensively from TT01, showing 13,000 point mutations, 330 frameshifts, and 220 strain-specific regions with a total length of more than 300kb in each of the compared genomes.Conclusion: sAccording to the major phenotypic and genotypic differences, the rifampicin resistant P. luminescens strain, now named strain DJC, has to be considered as an independent isolate rather than a derivative of strain TT01. Strains TT01 and DJC both belong to P. luminescens subsp. laumondii.
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