Development of a chemically defined medium for Paenibacillus polymyxa by parallel online monitoring of the respiration activity in microtiter plates.

BACKGROUND: One critical parameter in microbial cultivations is the composition of the cultivation medium. Nowadays, the application of chemically defined media increases, due to a more defined and reproducible fermentation performance than in complex media. In order, to improve cost-effectiveness of fermentation processes using chemically defined media, the media should not contain nutrients in large excess. Additionally, to obtain high product yields, the nutrient concentrations should not be limiting. Therefore, efficient medium optimization techniques are required which adapt medium compositions to the specific nutrient requirements of microorganisms. RESULTS: Since most Paenibacillus cultivation protocols so far described in literature are based on complex ingredients, in this study, a chemically defined medium for an industrially relevant Paenibacillus polymyxa strain was developed. A recently reported method, which combines a systematic experimental procedure in combination with online monitoring of the respiration activity, was applied and extended to identify growth limitations for Paenibacillus polymyxa. All cultivations were performed in microtiter plates. By systematically increasing the concentrations of different nutrient groups, nicotinic acid was identified as a growth-limiting component. Additionally, an insufficient buffer capacity was observed. After optimizing the growth in the chemically defined medium, the medium components were systematically reduced to contain only nutrients relevant for growth. Vitamins were reduced to nicotinic acid and biotin, and amino acids to methionine, histidine, proline, arginine, and glutamate. Nucleobases/-sides could be completely left out of the medium. Finally, the cultivation in the reduced medium was reproduced in a laboratory fermenter. CONCLUSION: In this study, a reliable and time-efficient high-throughput methodology was extended to investigate limitations in chemically defined media. The interpretation of online measured respiration activities agreed well with the growth performance of samples measured in parallel via offline analyses. Furthermore, the cultivation in microtiter plates was validated in a laboratory fermenter. The results underline the benefits of online monitoring of the respiration activity already in the early stages of process development, to avoid limitations of medium components, oxygen limitation and pH inhibition during the scale-up.

Insights in the Complex DegU, DegS, and Spo0A Regulation System of Paenibacillus polymyxa by CRISPR-Cas9-Based Targeted Point Mutations.

Despite being unicellular organisms, bacteria undergo complex regulation mechanisms which coordinate different physiological traits. Among others, DegU, DegS, and Spo0A are the pleiotropic proteins which govern various cellular responses and behaviors. However, the functions and regulatory networks between these three proteins are rarely described in the highly interesting bacterium Paenibacillus polymyxa. In this study, we investigate the roles of DegU, DegS, and Spo0A by introduction of targeted point mutations facilitated by a CRISPR-Cas9-based system. In total, five different mutant strains were generated, the single mutants DegU Q218*, DegS L99F, and Spo0A A257V, the double mutant DegU Q218* DegS L99F, and the triple mutant DegU Q218* DegS L99F Spo0A A257V. Characterization of the wild-type and the engineered strains revealed differences in swarming behavior, conjugation efficiency, sporulation, and viscosity formation of the culture broth. In particular, the double mutant DegU Q218* DegS L99F showed a significant increase in conjugation efficiency as well as a stable exopolysaccharides formation. Furthermore, we highlight similarities and differences in the roles of DegU, DegS, and Spo0A between P. polymyxa and related species. Finally, this study provides novel insights into the complex regulatory system of P. polymyxa DSM 365. IMPORTANCE To date, only limited knowledge is available on how complex cellular behaviors are regulated in P. polymyxa. In this study, we investigate several regulatory proteins which play a role in governing different physiological traits. Precise targeted point mutations were introduced to their respective genes by employing a highly efficient CRISPR-Cas9-based system. Characterization of the strains revealed some similarities, but also differences, to the model bacterium Bacillus subtilis with regard to the regulation of cellular behaviors. Furthermore, we identified several strains which have superior performance over the wild-type. The applicability of the CRISPR-Cas9 system as a robust genome editing tool, in combination with the engineered strain with increased genetic accessibility, would boost further research in P. polymyxa and support its utilization for biotechnological applications. Overall, our study provides novel insights, which will be of importance in understanding how multiple cellular processes are regulated in Paenibacillus species.

[Bovine trace mineral concentrations in different sample media with emphasis on fecal analysis]. / Konzentration von Spurenelementen beim Rind in verschiedenen Probenmedien unter besonderer Berücksichtigung von Kotproben.

OBJECTIVE: The objective of this study was to evaluate the suitability of feces as sample material for the evaluation of trace mineral supply in cattle. Fecal trace element concentrations of iron, copper, zinc, selenium, manganese and molybdenum were compared with those in feed. Furthermore, fecal trace element-concentrations were compared with those in various additional sample media (serum, plasma, whole blood, urine and hair). MATERIALS AND METHODS: Samples (feces, blood, urine, hair) from up to 10 cows in the close-up (3-0 weeks a. p.) and high-yielding (6-20 weeks p. p.) group each were collected during 73 farm visits on 63 dairy farms. Pooled samples of each lactation group were formed and the concentrations of trace elements were determined with ICP-OES or ICP-MS. Furthermore, TMR was analyzed. Additionally, feces from cattle in cow-calf herds during grazing and housing period were evaluated. RESULTS: Nutritional trace mineral requirements were exceeded by the majority of feeds. Trace mineral concentrations were 2- to 3-fold higher in feces than in feed. In this study, the strongest significant correlation existed between feed and fecal concentrations for all elements. Seasonal variations were detected for iron, copper and manganese. Furthermore, iron concentration differed depending on lactation stage. CONCLUSION AND CLINICAL RELEVANCE: Feed intake of the trace minerals analyzed is most optimally reflected by the fecal concentration in comparison to other substrates. Fecal analysis of these minerals may therefore be employed as an alternative to feed analysis with regards to the evaluation of nutritional trace mineral supply. Analyzing fecal trace mineral concentrations possesses practical significance wherever feed or TMR cannot be analyzed. In particular, this concerns grazing animals, heifers, mother cows and cattle in barns without TMR. Assessment of the complete metabolic status only on the basis of fecal contents is insufficient. However, subnormal fecal copper-, zinc- and manganese concentrations may be suggestive of an inadequate metabolic situation and represent an indication for further diagnostics.

Subtyping Of Triple Negative Breast Carcinoma On The Basis Of RTK Expression.

Background: "Triple-negative breast cancers" (TNBC) comprise a heterogeneous group of about 15% of invasive BCs lacking the expression of estrogen and progesterone receptors (ER, PR) and the expression of HER2 (ERBB2) and are therefore no established candidates for targeted treatment options in BC, i.e., endocrine and anti-HER2 therapy. The aim of the present study was to use gene expression profiling and immunohistochemical (IHC) characterization to identify receptor tyrosine kinase (RTK) profiles that would allow patient stratification for the purposes of target-oriented personalized tumor therapy in TNBC. Methods: Twenty-nine cases of TNBC selected according to routine diagnostic IHC/cytogenetic criteria were examined by reverse transcription polymerase chain reaction (RT-PCR). RTK mRNA expression profiles were generated for a total of 31 tumor-relevant biomarkers, mainly belonging to the IGF- and EGF-receptor families but also including biomarkers related to downstream signaling. Protein expression of selected biomarkers was investigated by IHC. Results: Hierarchical cluster analysis revealed a dichotomous differentiation pattern amongst TNBCs. A significant difference in gene expression was observed for 16 of the 31 RTK-associated tumor relevant biomarkers between the two newly identified TNBC subgroups. The findings were verified at the posttranslational level by the IHC data. The RTKs HER4, IGF-1R and IGF-2R and the hormone receptors ER and PR below the IHC detection limit play a central role in the differentiation of the two TNBC subgroups. Observed survival was reported as Kaplan-Meier estimates and point towards an improved survival of patients with RTK-high with superior three-year survival rate of 100% compared to RTK-low gene signatures with superior three-year survival rate of 60% (log-rank test, p-value = 0.022). Conclusion: Gene-expression and IHC analysis of the EGF and IGF receptor families and biomarkers associated with downstream signaling point to the existence of two distinct TNBC subtypes. The RTKs HER4, IGF-1R, IGF-2R and the hormone receptors ER and PR appear to be of particular importance here. Based on survival analysis the differentiation of TNBC with RTK-high and RTK-low gene signatures seems to be of prognostic relevance. Additionally, correlation analysis of the relationship between RTKs and ER suggests co-regulatory mechanisms that may have potential significance in new therapeutic approaches.

[Mineral concentrations in cattle in different sample media with emphasis on fecal analysis]. / Konzentrationen an Mengenelementen beim Rind in verschiedenen Probenmedien unter besonderer Berücksichtigung von Kotproben.

OBJECTIVE: The objective of this study was to evaluate whether the concentrations of calcium (Ca), phosphorus (P), magnesium (Mg), potassium (K), sodium (Na) and sulfur (S) in the feces reflect the content in feeds and whether analysis of the feces provides information on the nutritional supply of the body with these elements. MATERIALS AND METHODS: For this purpose, element concentrations in feed and in serum, plasma, whole blood, urine and hair were compared. During 73 farm visits on 63 dairy farms, samples were collected from up to 10 cows in both the close-up (3-0 weeks a. p.) and high-yielding (6th-20th week p. p.) groups. Concentrations of Ca, P, Mg, K, Na and S were determined in pooled samples of each lactation group and in a sample of the total mixed ration (TMR) by ICP-OES. Additionally, feces from beef cows during the grazing and housing periods were analyzed. RESULTS: Mineral concentrations in feed samples were sufficiently high to meet the nutritional requirements. Mineral concentrations were higher in feces than in feed, with the exception of Na and K. There were significant correlations between feed and fecal concentrations for all elements, whereby the correlations between the TMR and urine concentrations were higher for K, Na and S. Seasonal variations were detected in all elements, with the exception of Ca. Furthermore, Ca, P, Mg and S concentrations differed depending on the stage of lactation. CONCLUSION: The fecal concentration of Ca, P and Mg best reflect the intake with the feed in comparison to other substrates. Fecal analysis of these elements can, therefore, be applied as an alternative to feed analysis regarding the evaluation of the nutritional mineral supply. Assessment of the metabolic status only on the basis of the fecal contents is insufficient. The urine Mg concentration provides information regarding Mg metabolic status. Furthermore, urine is the substrate of choice for the evaluation of the K and Na metabolic status. The most useful information about S nutritional and metabolic status is provided by combining the results of fecal and urinary examinations.

Gene expression analysis in biomarker research and early drug development using function tested reverse transcription quantitative real-time PCR assays.

The identification of new biomarkers is essential in the implementation of personalized health care strategies that offer new therapeutic approaches with optimized and individualized treatment. In support of hypothesis generation and testing in the course of our biomarker research an online portal and respective function-tested reverse transcription quantitative real-time PCR assays (RT-qPCR) facilitated the selection of relevant biomarker genes. We have established workflows applicable for convenient high throughput gene expression analysis in biomarker research with cell lines (in vitro studies) and xenograft mouse models (in vivo studies) as well as formalin-fixed paraffin-embedded tissue (FFPET) sections from various human research and clinical tumor samples. Out of 92 putative biomarker candidate genes selected in silico, 35 were shown to exhibit differential expression in various tumor cell lines. These were further analysed by in vivo xenograft mouse models, which identified 13 candidate genes including potential response prediction biomarkers and a potential pharmacodynamic biomarker. Six of these candidate genes were selected for further evaluation in FFPET samples, where optimized RNA isolation, reverse transcription and qPCR assays provided reliable determination of relative expression levels as precondition for differential gene expression analysis of FFPET samples derived from projected clinical studies. Thus, we successfully applied function tested RT-qPCR assays in our biomarker research for hypothesis generation with in vitro and in vivo models as well as for hypothesis testing with human FFPET samples. Hence, appropriate function-tested RT-qPCR assays are available in biomarker research accompanying the different stages of drug development, starting from target identification up to early clinical development. The workflow presented here supports the identification and validation of new biomarkers and may lead to advances in efforts to achieve the goal of personalized health care.

Metabolic flux engineering of L-lysine production in Corynebacterium glutamicum--over expression and modification of G6P dehydrogenase.

In the present work, metabolic flux engineering of Corynebacterium glutamicum was carried out to increase lysine production. The strategy focused on engineering of the pentose phosphate pathway (PPP) flux by different genetic modifications. Over expression of the zwf gene, encoding G6P dehydrogenase, in the feedback-deregulated lysine-producing strain C. glutamicum ATCC 13032 lysC(fbr) resulted in increased lysine production on different carbon sources including the two major industrial sugars, glucose and sucrose. The additional introduction of the A243T mutation into the zwf gene and the over expression of fructose 1,6-bisphosphatase resulted in a further successive improvement of lysine production. Hereby the point mutation resulted in higher affinity of G6P dehydrogenase towards NADP and reduced sensitivity against inhibition by ATP, PEP and FBP. Overall, the lysine yield increased up to 70% through the combination of the different genetic modifications. Through strain engineering formation of trehalose was reduced by up to 70% due to reduced availability of its precursor G6P. Metabolic flux analysis revealed a 15% increase of PPP flux in response to over expression of the zwf gene. Overall a strong apparent NADPH excess resulted. Redox balancing indicated that this excess is completely oxidized by malic enzyme.

Genome-wide analysis of mRNAs regulated by the THO complex in Drosophila melanogaster.

In yeast cells, the THO complex has been implicated in mitotic recombination, transcription elongation and mRNA nuclear export. The stable core of THO consists of Tho2p, Hpr1p, Mft1p and Thp2p. Whether a complex with similar functions assembles in metazoa has not yet been established. Here we report that Drosophila melanogaster THO consists of THO2, HPR1 and three proteins, THOC5-THOC7, which have no orthologs in budding yeast. Gene expression profiling in cells depleted of THO components revealed that <20% of the transcriptome was regulated by THO. Nonetheless, export of heat-shock mRNAs under heat stress was strictly dependent on THO function. Notably, 8% of upregulated genes encode proteins involved in DNA repair. Thus, although THO function seems to be conserved, the vast majority of mRNAs are transcribed and exported independently of THO in D. melanogaster.

RanBP2/Nup358 provides a major binding site for NXF1-p15 dimers at the nuclear pore complex and functions in nuclear mRNA export.

Metazoan NXF1-p15 heterodimers promote the nuclear export of bulk mRNA across nuclear pore complexes (NPCs). In vitro, NXF1-p15 forms a stable complex with the nucleoporin RanBP2/Nup358, a component of the cytoplasmic filaments of the NPC, suggesting a role for this nucleoporin in mRNA export. We show that depletion of RanBP2 from Drosophila cells inhibits proliferation and mRNA export. Concomitantly, the localization of NXF1 at the NPC is strongly reduced and a significant fraction of this normally nuclear protein is detected in the cytoplasm. Under the same conditions, the steady-state subcellular localization of other nuclear or cytoplasmic proteins and CRM1-mediated protein export are not detectably affected, indicating that the release of NXF1 into the cytoplasm and the inhibition of mRNA export are not due to a general defect in NPC function. The specific role of RanBP2 in the recruitment of NXF1 to the NPC is highlighted by the observation that depletion of CAN/Nup214 also inhibits cell proliferation and mRNA export but does not affect NXF1 localization. Our results indicate that RanBP2 provides a major binding site for NXF1 at the cytoplasmic filaments of the NPC, thereby restricting its diffusion in the cytoplasm after NPC translocation. In RanBP2-depleted cells, NXF1 diffuses freely through the cytoplasm. Consequently, the nuclear levels of the protein decrease and export of bulk mRNA is impaired.

Genome-wide analysis of nuclear mRNA export pathways in Drosophila.

NXF1, p15 and UAP56 are essential nuclear mRNA export factors. The fraction of mRNAs exported by these proteins or via alternative pathways is unknown. We have analyzed the relative abundance of nearly half of the Drosophila transcriptome in the cytoplasm of cells treated with the CRM1 inhibitor leptomycin-B (LMB) or depleted of export factors by RNA interference. While the vast majority of mRNAs were unaffected by LMB, the levels of most mRNAs were significantly reduced in cells depleted of NXF1, p15 or UAP56. The striking similarities of the mRNA expression profiles in NXF1, p15 and UAP56 knockdowns show that these proteins act in the same pathway. The broad effect on mRNA levels observed in these cells indicates that the functioning of this pathway is required for export of most mRNAs. Nonetheless, a set of mRNAs whose export was unaffected by the depletions and some requiring NXF1:p15 but not UAP56 were identified. In addition, our analysis revealed a feedback loop by which a block to mRNA export triggers the upregulation of genes involved in this process.

Cdc37 is essential for chromosome segregation and cytokinesis in higher eukaryotes.

Cdc37 has been shown to be required for the activity and stability of protein kinases that regulate different stages of cell cycle progression. However, little is known so far regarding interactions of Cdc37 with kinases that play a role in cell division. Here we show that the loss of function of Cdc37 in Drosophila leads to defects in mitosis and male meiosis, and that these phenotypes closely resemble those brought about by the inactivation of Aurora B. We provide evidence that Aurora B interacts with and requires the Cdc37/Hsp90 complex for its stability. We conclude that the Cdc37/Hsp90 complex modulates the function of Aurora B and that most of the phenotypes brought about by the loss of Cdc37 function can be explained by the inactivation of this kinase. These observations substantiate the role of Cdc37 as an upstream regulatory element of key cell cycle kinases.

Nuclear export of mRNA by TAP/NXF1 requires two nucleoporin-binding sites but not p15.

Metazoan NXF1/p15 heterodimers promote export of bulk mRNA through nuclear pore complexes (NPC). NXF1 interacts with the NPC via two distinct structural domains, the UBA-like domain and the NTF2-like scaffold, which results from the heterodimerization of the NTF2-like domain of NXF1 with p15. Both domains feature a single nucleoporin-binding site, and they act synergistically to promote NPC translocation. Whether the NTF2-like scaffold (and thereby p15) contributes only to NXF1/NPC association or is also required for other functions, e.g., to impart directionality to the export process by regulating NXF1/NPC or NXF1/cargo interactions, remains unresolved. Here we show that a minimum of two nucleoporin-binding sites is required for NXF1-mediated export of cellular mRNA. These binding sites can be provided by an NTF2-like scaffold followed by a UBA-like domain (as in the wild-type protein) or by two NTF2-like scaffolds or two UBA-like domains in tandem. In the latter case, the export activity of NXF1 is independent of p15. Thus, as for the UBA-like domain, the function of the NTF2-like scaffold is confined to nucleoporin binding. More importantly, two copies of either of these domains are sufficient to promote directional transport of mRNA cargoes across the NPC.