Amino acid transport in Penicillium chrysogenum in relation to precursor supply for beta-lactam production

Penicilline wordt in de industrie gewonnen uit de schimmel Penicillium chrysogenum. In de cellen van deze schimmel vormt het antibioticum zich in enkele stappen. De eerste daarvan is het aaneenkoppelen van drie aminozuurmoleculen, waaronder alfa-aminoadipaat. Die aminozuren worden alledrie door de cellen zelf geproduceerd en hoeven voor de penicillineproductie in principe dus niet 'gevoed' te worden. Maar als men wel alfa-aminoadipaat toevoegt aan het groeimedium, gaat de penicillineproductie omhoog. Dit betekent in elk geval dat alfa-aminoadipaat wordt opgenomen door de cellen. Kern van het promotieonderzoek van Hein Trip is de vraag: hoe wordt het van buiten naar binnen getransporteerd? Celmembranen bevatten verschillende eiwitten die aminozuren kunnen transporteren. In het onderzoek karakteriseerde Trip vier van dit soort eiwitten uit Penicillium chrysogenu. Twee daarvan bleken in staat om alfa-aminoadipaat te transporteren over een celmembraan.

Improved Acid Stress Survival of Lactococcus lactis Expressing the Histidine Decarboxylation Pathway of Streptococcus thermophilus CHCC1524

Background: Degradative amino acid decarboxylation pathways in bacteria have diverse physiological functions. Results: A histidine decarboxylation pathway introduced in L. lactis improves acid stress survival, and synergy with the glycolytic pathway is demonstrated. Conclusion: The physiological benefit of the new pathway is strongly dependent on the properties of the host organism. Significance: Acquisition of the histidine decarboxylation pathway mimics successful horizontal gene transfer

Regulation of ykrL (htpX) by Rok and YkrK, a Novel Type of Regulator in Bacillus subtilis

Expression of ykrL of Bacillus subtilis, encoding a close homologue of the Escherichia coli membrane protein quality control protease HtpX, was shown to be upregulated under membrane protein overproduction stress. Using DNA affinity chromatography, two proteins were found to bind to the promoter region of ykrL: Rok, known as a repressor of competence and genes for extracytoplasmic functions, and YkrK, a novel type of regulator encoded by the gene adjacent to ykrL but divergently transcribed. Electrophoretic mobility shift assays showed Rok and YkrK binding to the ykrL promoter region as well as YkrK binding to the ykrK promoter region. Comparative bioinformatic analysis of the ykrL promoter regions in related Bacillus species revealed a consensus motif, which was demonstrated to be the binding site of YkrK. Deletion of rok and ykrK in a PykrL-gfp reporter strain showed that both proteins are repressors of ykrL expression. In addition, conditions which activated PykrL (membrane protein overproduction, dissipation of the membrane potential, and salt and phenol stress) point to the involvement of YkrL in membrane protein quality control

Evidence of Two Functionally Distinct Ornithine Decarboxylation Systems in Lactic Acid Bacteria

Biogenic amines are low-molecular-weight organic bases whose presence in food can result in health problems. The biosynthesis of biogenic amines in fermented foods mostly proceeds through amino acid decarboxylation carried out by lactic acid bacteria (LAB), but not all systems leading to biogenic amine production by LAB have been thoroughly characterized. Here, putative ornithine decarboxylation pathways consisting of a putative ornithine decarboxylase and an amino acid transporter were identified in LAB by strain collection screening and database searches. The decarboxylases were produced in heterologous hosts and purified and characterized in vitro, whereas transporters were heterologously expressed in Lactococcus lactis and functionally characterized in vivo. Amino acid decarboxylation by whole cells of the original hosts was determined as well. We concluded that two distinct types of ornithine decarboxylation systems exist in LAB. One is composed of an ornithine decarboxylase coupled to an ornithine/putrescine transmembrane exchanger. Their combined activities results in the extracellular release of putrescine. This typical amino acid decarboxylation system is present in only a few LAB strains and may contribute to metabolic energy production and/or pH homeostasis. The second system is widespread among LAB. It is composed of a decarboxylase active on ornithine and L-2,4-diaminobutyric acid (DABA) and a transporter that mediates unidirectional transport of ornithine into the cytoplasm. Diamines that result from this second system are retained within the cytosol.

Prognostic value of myocardial perfusion scintigraphy in type 2 diabetic patients with mild, stable angina pectoris

Aim: To determine the prognostic value of reversible myocardial perfusion defects on myocardial perfusion scintigraphy (MPS) in patients with type 2 diabetes mellitus and mild anginal complaints. Methods and results: In the MERIDIAN trial, patients with diabetes mellitus type 2, stable, mild anginal symptoms (Canadian Cardiovascular Society classification (CCS) I-II/IV) and reversible perfusion defects were randomized to either continued pharmacological treatment or early invasive treatment. In this sub analysis, the severity of the myocardial perfusion defect was related to the occurrence of cardiac death and non-fatal myocardial infarction, in 319 patients (63% male, 65 ± 9 years). During follow-up (2.2 ± 0.6 years), 14 patients had a cardiac event: 3 in 171 patients without myocardial ischemia and 11 in 148 patients with myocardial ischemia. Annual event rates rose from 0.8% to 5.8% with increasing severity of myocardial ischemia. Multivariable analysis identified the presence of severe myocardial ischemia (hazard ratio (HR) 5.45, 95%CI 1.89-15.71) and insulin use (HR 4.00, 95%CI 1.25-12.75) as independent predictors of cardiac events. Conclusions: Type 2 diabetics with mild anginal symptoms with no or moderate myocardial ischemia have a low annual cardiac event rate. In patients with severe myocardial ischemia event rate increased 3-6 fold

Spatial separation of FtsZ and FtsN during cell division

The division of Escherichia coli is mediated by a collection of some 34 different proteins that are recruited to the division septum and are thought to assemble into a macromolecular complex known as ‘the divisome’. Herein, we have endeavored to better understand the structure of the divisome by imaging two of its core components; FtsZ and FtsN. Super resolution microscopy (SIM and gSTED) indicated that both proteins are localized in large assemblies, which are distributed around the division septum (i.e., forming a discontinuous ring). Although the rings had similar radii prior to constriction, the individual densities were often spatially separated circumferentially. As the cell envelope constricted, the discontinuous ring formed by FtsZ moved inside the discontinuous ring formed by FtsN. The radial and circumferential separation observed in our images indicates that the majority of FtsZ and FtsN molecules are organized in different macromolecular assemblies, rather than in a large super‐complex. This conclusion was supported by fluorescence recovery after photobleaching measurements, which indicated that the dynamic behavior of the two macromolecular assemblies was also fundamentally different. Taken together, the data indicates that constriction of the cell envelope is brought about by (at least) two spatially separated complexes

PcMtr, an aromatic and neutral aliphatic amino acid permease of Penicillium chrysogenum

The gene encoding an aromatic and neutral aliphatic amino acid permease of Penicillium chrysogenum was cloned, functionally expressed and characterized in Saccharomyces cerevisiae M4276. The permease, designated PcMtr, is structurally and functionally homologous to Mtr of Neurospora crassa, and unrelated to the Amino Acid Permease (AAP) family which includes most amino acid permeases in fungi. Database searches of completed fungal genome sequences reveal that Mtr type permeases are not widely distributed among fungi, suggesting a specialized function.