A "second-generation" production strain was derived from a Corynebacterium glutamicum pantothenate producer by rational design to assess its potential to synthesize and accumulate the vitamin ...pantothenate by batch cultivation. The new pantothenate production strain carries a deletion of the ilvA gene to abolish isoleucine synthesis, the promoter down-mutation P-ilvEM3 to attenuate ilvE gene expression and thereby increase ketoisovalerate availability, and two compatible plasmids to overexpress the ilvBNCD genes and duplicated copies of the panBC operon. Production assays in shake flasks revealed that the P-ilvEM3 mutation and the duplication of the panBC operon had cumulative effects on pantothenate production. During pH-regulated batch cultivation, accumulation of 8 mM pantothenate was achieved, which is the highest value reported for C. glutamicum. Metabolic flux analysis during the fermentation demonstrated that the P-ilvEM3 mutation successfully reoriented the carbon flux towards pantothenate biosynthesis. Despite this repartition of the carbon flux, ketoisovalerate not converted to pantothenate was excreted by the cell and dissipated as by-products (ketoisocaproate, DL-2,3,-dihydroxy-isovalerate, ketopantoate, pantoate), which are indicative of saturation of the pantothenate biosynthetic pathway. Genome-wide expression analysis of the production strain during batch cultivation was performed by whole-genome DNA microarray hybridization and agglomerative hierarchical clustering, which detected the enhanced expression of genes involved in leucine biosynthesis, in serine and glycine formation, in regeneration of methylenetetrahydrofolate, in de novo synthesis of nicotinic acid mononucleotide, and in a complete pathway of acyl coenzyme A conversion. Our strategy not only successfully improved pantothenate production by genetically modified C. glutamicum strains but also revealed new constraints in attaining high productivity.
1 INBIOTEC (Instituto de Biotecnología de León), Parque Científico de León, Avda. Real, 1, 24006 León, Spain
2 Institut für Genomforschung und Systembiologie, Centrum für Biotechnologie, Universität ...Bielefeld, Universitätsstrasse 25, 33615 Bielefeld, Germany
3 Área de Microbiología, Departamento de Biología Molecular, Universidad de León, Campus de Vegazana, s/n. 24071 León, Spain
Correspondence Juan F. Martín jf.martin{at}unileon.es
Genome-wide transcription profile analysis of the heat-shocked wild-type strain under moderate (40 °C) and severe heat stress (50 °C) revealed that a large number of genes are differentially expressed after heat shock. Of these, 358 genes were upregulated and 420 were downregulated in response to moderate heat shock (40 °C) in Corynebacterium glutamicum . Our results confirmed the HrcA/controlling inverted repeat of chaperone expression (CIRCE)-dependent and HspR/HspR-associated inverted repeat (HAIR)-dependent upregulation of chaperones following heat shock. Other genes, including clusters of orthologous groups (COG) related to macromolecule biosynthesis and several transcriptional regulators (COG class K), were upregulated, explaining the large number of genes affected by heat shock. Mutants having deletions in the hrcA or hspR regulators were constructed, which allowed the complete identification of the genes controlled by those systems. The up- or downregulation of several genes observed in the microarray experiments was validated by Northern blot analyses and quantitative (real-time) reverse-transcription PCR. These analyses showed a heat-shock intensity-dependent response (differential response) in the HspR/HAIR system, in contrast to the non-differential response shown by the HrcA/CIRCE-regulated genes.
Abbreviations: CIRCE, controlling inverted repeat of chaperone expression; COG, clusters of orthologous groups; CtsR, class three stress gene repressor; HAIR, HspR-associated inverted repeat; q-RT-PCR, quantitative (real-time) reverse-transcription PCR; ROSE, repression of heat-shock gene expression
The array data discussed in this paper have been deposited in EBI and are accessible through ArrayExpress series accession number E-MTAB-66.
Tables of microarray data are available as supplementary material with the online version of this paper.
Microarray analysis has become a popular and routine method in functional genomics. It is typical for such experiments to involve a small number of replicates, which causes unreliable estimates of ...the sample variance. Microarrays have fostered the development of new statistical methods to analyze data resulting from experiments with small sample sizes. In this study, we tackle the problem of evaluating the performance of statistical tests for generating ranked gene lists from two-channel direct comparisons. We propose an evaluation method based on a oligonucleotide microarray with a large number of replicate spots yielding a maximum of 400 replicates per gene. We apply Spearman’s rank correlation coefficient to ranked gene-lists generated by eight widely used microarray specific test statistics, which are applied to small random samples. We could show that variance stabilizing methods such as Cyber-T, SAM, and LIMMA can be beneficial for very small sample sizes and that SAM and the
t-test provide stronger control of the type I error rate than the other methods. Specifically, we report that for four replicates all methods reach a high to very high correlation with our reference standard.
In the upper Hoanib River catchment area, northwestern Namibia, fine-grained silty deposits are widespread. Accumulated as river-end deposits they form excellent geomorphological archives of a highly ...sensitive desert-margin area. Different sedimentary complexes are separated sedimentologically and by luminescence dating. The deposits give evidence of climate oscillations at the eastern margin of the Namib Desert during the past 50,000 years.
In northwestern Namibia, the dry conditions of the desert-margin area with less than 200–300 mm mean annual precipitation prevailed at ∼60–40 ka and at ∼34–24 ka when sediment complex-I accumulated. During the Last Glacial Maximum (LGM), fluvial activity and sedimentation were most likely interrupted due to arid conditions. Between ∼12 ka and the Mid-Holocene higher rainfall and runoff led to the aggradation of sediment complex-II. The climate was more humid than before the LGM, but drier than at present. After ∼3 ka runoff increased and the Hoanib River re-eroded the older deposits forming deep channels. A prominent, more sandy and gravely 4 m-terrace in the upper and middle course of the Hoanib River dates to the Little Ice Age (LIA). It is likely that this terrace is genetically linked with the Amspoort Silt formation in the Lower Hoanib valley.
Summary
Background
We investigated the applicability of both Acute Physiology And Chronic Health Evaluation II (APACHE II) and Mannheim Peritonitis Index (MPI) in transplant recipients with ...intestinal perforation as polymicrobial peritonitis is highly life-threatening in patients with impaired immunological defence and the course of abdominal sepsis is aberrant compared to non-immunosuppressed individuals.
Methods
In a retrospective database analysis, we validated the consistency of APACHE II and MPI in death prediction in septic peritonitis. In a matched-pair analysis we then investigated their applicability in immunosuppressed transplant recipients. The validation of the scores was performed with 125 non-immunosuppressed patients. In a matched-pair analysis 16 immunosuppressed transplant recipients were compared with 32 controls.
Results
In accordance with clinical observations, statistical analyses revealed significantly higher MPI scores in immunosuppressed transplant recipients (
P
= 0.0007), whereas APACHE II scores were comparable. Linear regression analysis revealed that predicted death rates derived from APACHE II and MPI differ significantly (
P
= 0.0344) in immunosuppressed transplant recipients compared to controls.
Conclusions
Both scores consistently serve as reliable tools for prediction of lethality in septic peritonitis but they fail in immunosuppressed organ recipients. Our findings clearly confirm that in immunosuppressed patients severe peritonitis might be present despite an a- or oligosymptomatic clinical course.
Detailed geomorphological and chronological investigations of the NW-Namibian Amspoort Silt formation show that the sediments are typical river-end deposits. This type of endoreic sediment, occuring ...only in desert margin areas, provides valuable information about the palaeo-environment. In the Hoanib valley, the fine-grained deposits have buried riverine trees. Radiocarbon dating of the wood and luminescence dating of the sediments allow a detailed reconstruction of the aggradation processes. Accumulation started ∼10 km downstream of Amspoort around the beginning of the 15th century and ended in the 19th century, some kilometres upstream of Amspoort. This upstream shift of sedimentation during the Little Ice Age was caused by gradually decreasing runoff resulting from aridification of the upper part of the Hoanib river catchment lying east of the Namib desert margin ≥1.200 m a.s.l.
The Amspoort Silt terrace is evidence of palaeo-hydrological fluctuations in NW-Namibia. At present, the Hoanib river erodes deeply into the silty deposits, indicating that NW-Namibia receives more monsoonal rainfall today than during the Little Ice Age. However, this contradicts the hypothesis of a (continual) natural aridification of NW-Namibia (Damaraland, Kaokoveld) since the mid-19th century in the course of global climatic change. Rather, deposition and erosion of the Amspoort Silts indicate that landscape degradation in NW-Namibia is primarily anthropogenically induced and most probably not accelerated by a decrease in precipitation.
Abstract Objective Orthotopic liver transplantation (OLT) in rats is frequently used as an experimental model. Numerous surgical techniques have been developed that enable the investigator to conduct ...clinically relevant studies. The objective of this study was to develop a rat model of acute and chronic rejection, to explicitly study technical modifications of vascular anastomoses with precision, and to examine histopathologic and functional changes in the graft. Materials and Methods With DA-(RT1av1) rats as donors and Lewis-(RT1) rats as recipients, arterialized OLT was performed using a combined suture, cuff, and splint method. Recipients were divided into 5 groups: syngeneic control rats (group 1), allogeneic control rats (group 2), allogeneic OLT rats with low-dose tacrolimus (FK506) immunosuppression (group 3), allogeneic OLT rats with high-dose tacrolimus immunosuppression (group 4), and allogeneic OLT rats with high-dose tacrolimus immunosuppression and retrograde reperfusion via the infrahepatic caval vein (group 5). After OLT, serum parameters were determined and hepatic biopsy specimens were sampled. We examined the effects of acute rejection with or without immunosuppression therapy at histopathologic evaluation. Results Liver grafts in syngeneic and allogeneic rats (groups 1, 2, 4, and 5) demonstrated normal serum parameters and histopathologic findings at 10 days after OLT, and 93% survival at 3 months. The simplified technique using 1 suture and 2 cuff anastomoses provided the best short- and long-term survival after OLT in all groups. Retrograde perfusion via the infrahepatic caval vein resulted in lower postoperative liver enzyme values. Conclusion The present model is feasible, enabling comprehensive preclinical experimental research on liver transplantation. Furthermore, we provide helpful instructions for learning this surgical technique.
Summary
In a recent study, the putative regulatory gene cg0012 was shown to belong to the regulon of McbR, a global transcriptional regulator of sulphur metabolism in Corynebacterium glutamicum ATCC ...13032. A deletion of cg0012, now designated ssuR (sulphonate sulphur utilization regulator), led to the mutant strain C. glutamicum DK100, which was shown to be blocked in the utilization of sulphonates as sulphur sources. According to DNA microarray hybridizations, transcription of the ssu and seu genes, encoding the sulphonate utilization system of C. glutamicum, was considerably decreased in C. glutamicum DK100 when compared with the wild‐type strain. Electrophoretic mobility shift assays with purified SsuR protein demonstrated that the upstream regions of ssuI, seuABC, ssuD2 and ssuD1CBA contain SsuR binding sites. A nucleotide sequence alignment of the four DNA fragments containing the SsuR binding sites revealed a common 21 bp motif consisting of T‐, GC‐ and A‐rich domains. Mapping of the transcriptional start sites in front of ssuI, seuABC, ssuD2 and ssuD1CBA indicated that the SsuR binding sites are located directly upstream of identified promoter sequences and that the ssu genes are expressed by leaderless transcripts. Binding of the SsuR protein to its operator was shown to be diminished in vitro by the effector substance sulphate and its direct assimilation products adenosine 5′‐phosphosulphate, sulphite and sulphide. Real‐time reverse transcription polymerase chain reaction experiments verified that the expression of the ssu and seu genes was also repressed in vivo by the presence of sulphate or sulphite. Therefore, the regulatory protein SsuR activates the expression of the ssu and seu genes in C. glutamicum in the absence of the preferred sulphur source sulphate.
Chronic graft rejection mediated by cellular immune responses still poses a serious clinical problem in transplant surgery. Chemokines coordinate the recruitment of leukocytes in inflammatory and ...immune responses. Their precise functions in the rejection of allografts are still ill defined. This study investigates the role of chemokine receptor 4 (CCR4) in acute and chronic cardiac allograft rejection in mice. Allogeneic hearts were transplanted into CCR4 deficient (CCR4–/–) and control recipients. Reverse transcription‐PCR showed transcription of macrophage‐derived chemokine and thymus and activation‐regulated chemokine, the cognate chemokine ligands of CCR4, within the graft. Compared to wild‐type controls, acute allograft rejection in CCR4–/– recipients was only slightly prolonged. In contrast, in a gallium nitrate chronic cardiac allograft rejection model, cardiac graft survival was significantly prolonged in CCR4–/– recipients. A relative increase in the percentage of graft infiltrating CD8+ T cells in CCR4–/– recipients was observed 30 days after transplantation and was accompanied by a decrease in CD4+ T cells. Moreover, the percentage of NK1.1+CD3+ graft‐infiltrating cells was significantly reduced on day 5 and day 30 post transplantation. These findings indicate that CCR4 is involved in the recruitment of NK1.1+CD3+ cells into cardiac allografts and clearly establish an important and novel role for CCR4 in chronic graft rejection.
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