Live-cell imaging of Streptomyces conjugation.

Time-lapse imaging of conjugative plasmid transfer in Streptomyces revealed intriguing insights into the unique two-step conjugation process of this Gram+ mycelial soil bacterium. Differentially labelling of donor and recipient strains with distinct fluorescent proteins allowed the visualization of plasmid transfer in living mycelium. In nearly all observed matings, plasmid transfer occurred when donor and recipient hyphae made intimate contact at the lateral walls. Plasmid transfer does not involve a complete fusion of donor and recipient hyphae, but depends on a pore formed by the FtsK-like DNA translocase TraB. Following the initial transfer at the contact site of donor and recipient, the plasmids spread within the recipient mycelium by invading neighboring compartments, separated by cross walls. Intra-mycelial plasmid spreading depends on a septal cross wall localized multi-protein DNA translocation apparatus consisting of TraB and several Spd proteins and is abolished in a spd mutant. The ability to spread within the recipient mycelium is a crucial adaptation to the mycelial life style of Streptomyces, potentiating the efficiency of plasmid transfer.

Role of the Streptomyces spore wall synthesizing complex SSSC in differentiation of Streptomyces coelicolor A3(2).

A crucial stage of the Streptomyces life cycle is the sporulation septation, a process were dozens of cross walls are synchronously formed in the aerial hyphae in a highly coordinated manner. This process includes the remodeling of the spore envelopes to make Streptomyces spores resistant to detrimental environmental conditions. Sporulation septation and the synthesis of the thickened spore envelope in S. coelicolor A3(2) involves the Streptomyces spore wall synthesizing complex SSSC. The SSSC is a multi-protein complex including proteins directing peptidoglycan synthesis (MreBCD, PBP2, Sfr, RodZ) and cell wall glycopolymer synthesis (PdtA). It also includes two eukaryotic like serin/threonine protein kinases (eSTPK), PkaI and PkaH, which were shown to phosphorylate MreC. Since unbalancing phosphorylation activity by either deleting eSTPK genes or by expressing a second copy of an eSTPK gene affected proper sporulation, a model was developed, in which the activity of the SSSC is controlled by protein phosphorylation.

Conjugative DNA-transfer in Streptomyces, a mycelial organism.

Conjugative DNA-transfer in the Gram-positive mycelial soil bacterium Streptomyces, well known for the production of numerous antibiotics, is a unique process involving the transfer of a double-stranded DNA molecule. Apparently it does not depend on a type IV secretion system but resembles the segregation of chromosomes during bacterial cell division. A single plasmid-encoded protein, TraB, directs the transfer from the plasmid-carrying donor to the recipient. TraB is a FtsK-like DNA-translocase, which recognizes a specific plasmid sequence, clt, via interaction with specific 8-bp repeats. Chromosomal markers are mobilized by the recognition of clt-like sequences randomly distributed all over the Streptomyces chromosomes. Fluorescence microcopy with conjugative reporter plasmids and differentially labelled recipient strains revealed conjugative plasmid transfer at the lateral walls of the hyphae, when getting in contact. Subsequently, the newly transferred plasmids cross septal cross walls, which occur at irregular distances in the mycelium and invade the neighboring compartments, thus efficiently colonizing the recipient mycelium. This intramycelial plasmid spreading requires the DNA-translocase TraB and a complex of several Spd proteins. Inactivation of a single spd gene interferes with intramycelial plasmid spreading. The molecular function of the Spd proteins is widely unknown. Spd proteins of different plasmids are highly diverse, none showing sequence similarity to a functionally characterized protein. The integral membrane protein SpdB2 binds DNA, peptidoglycan and forms membrane pores in vivo and in vitro. Intramycelial plasmid spreading is an adaptation to the mycelial growth characteristics of Streptomyces and ensures the rapid dissemination of the plasmid within the recipient colony before the onset of sporulation.

Leiomyosarcoma of the inferior vena cava extending into the right atrium. A rare differential diagnosis of a right atrial tumor with fatal outcome.

A 54-year-old female patient presented with a progressive and deteriorating dyspnea at the slightest exertion in particular during the past few days before presentation. Transthoracic echocardiography revealed a large space-occupying lesion in the right atrium extending into the inferior vena cava (IVC). Abdominal magnetic resonance aortography showed an elongated space-occupying lesion in the IVC with a significant portion of the tumor and almost completely filling the right atrium accompanied by an infiltration of the hepatic and renal veins. A pronounced tumor infiltration of the IVC at the level of the liver was confirmed intraoperatively and immunohistochemical analysis showed a moderate to poorly differentiated leiomyosarcoma. The extended tumor was successfully removed by a complex operation of the thorax and abdomen but the procedure was accompanied by severe bleeding. A few hours following the procedure the patient died due to a further episode of irreversible intra-abdominal hemorrhage.

A single adenosine with a neutral pKa in the ribosomal peptidyl transferase center.

Biochemical and crystallographic evidence suggests that 23S ribosomal RNA (rRNA) is the catalyst of peptide bond formation. To explore the mechanism of this reaction, we screened for nucleotides in Escherichia coli 23S rRNA that may have a perturbed pKa (where Ka is the acid constant) based on the pH dependence of dimethylsulfate modification. A single universally conserved A (number 2451) within the central loop of domain V has a near neutral pKa of 7.6 +/- 0.2, which is about the same as that reported for the peptidyl transferase reaction. In vivo mutational analysis of this nucleotide indicates that it has an essential role in ribosomal function. These results are consistent with a mechanism wherein the nucleotide base of A2451 serves as a general acid base during peptide bond formation.

Hopanoids are formed during transition from substrate to aerial hyphae in Streptomyces coelicolor A3(2).

Streptomyces coelicolor A3(2) contains a cluster of putative isoprenoid and hopanoid biosynthetic genes. The strain does not produce the pentacyclic hopanoids in liquid culture but produces them on solid medium when sporulating. Mutants defective in the formation of aerial mycelium and spores (bld), with the exception of bldB, do not synthesize hopanoids, whereas mutants, which form aerial mycelium but no spores (whi), do. The membrane condensing hopanoids possibly may alleviate stress in aerial mycelium by diminishing water permeability across the membrane.

Local drug delivery via transvascular injection.

BACKGROUND: In this experimental series we tested drug distribution and systemic leakage using local drug delivery with a new transvascular injection system. METHODS: Porcine femoral and carotid arteries (n = 56) underwent local drug application with a new 5 French (Fr) over-the-wire needle-injection catheter system (NIC) using three needles. A radioactive indicator [C14-Carvedilol, 2.0 milliliter (ml); 0.03 milligram (mg)] was injected in two carotid and two femoral vessels in parallel. Serial blood withdrawal was performed thereafter. After randomization to different explantation times, the vessels, perivascular tissue, liver and spleen were removed [0.5, 1, 1.5, 3 and 4 hours after injection, respectively]. Radioactivity was determined in a scintillation counter or with autoradiography. The indicator amount was calculated in relation to total drug amount (100%). RESULTS: Use of the NIC caused vessel texture alteration in non-diseased porcine vessels, seen as vessel wall penetration and perivascular edema. After single injection the maximum of the indicator was found in perivascular tissue 0.5 hours at the application site (carotid perivascular tissue: 7.48%; femoral perivascular tissue: 2.56%). Thereafter, radioactivity in the artery increased and perivascular content declined. The maximum in femoral arteries (1 hour; 1.96%) occurred earlier and was significantly lower compared to carotid arteries (2 hours; 7.75%). Four hours post-injection, 1.4% of total drug amount was detectable in the carotid arteries and 0.6% was detected in the femoral arteries. Systemic content was measured after C14-Carvedilol application with a maximum in serum of 28% (10 minutes), liver 30% (0.5 hour) and spleen 0.6% (0.5 hour). After 3 hours, still 5% of the indicator was still measureable in the serum and liver and less than 0.1% was measurable in the spleen. LDD with the NIC system is dependent on the vascular anatomy. The data indicate redistribution from perivascular to vascular space thus allowing a prolonged vascular and perivascular drug delivery. The amount deliverable is lower than expected due to substantial systemic drug contamination with this catheter.

The ABC transporter Tba of Amycolatopsis balhimycina is required for efficient export of the glycopeptide antibiotic balhimycin.

All known gene clusters for glycopeptide antibiotic biosynthesis contain a conserved gene supposed to encode an ABC-transporter. In the balhimycin-producer Amycolatopsis balhimycina this gene (tba) is localised between the prephenate dehydrogenase gene pdh and the peptide synthetase gene bpsA. Inactivation of tba in A. balhimycina by gene replacement did not interfere with growth and did not affect balhimycin resistance. However, in the supernatant of the tba mutant RM43 less balhimycin was accumulated compared to the wild type; and the intra-cellular balhimycin concentration was ten times higher in the tba mutant RM43 than in the wild type. These data suggest that the ABC transporter encoded in the balhimycin biosynthesis gene cluster is not involved in resistance but is required for the efficient export of the antibiotic. To elucidate the activity of Tba it was heterologously expressed in Escherichia coli with an N-terminal His-tag and purified by nickel chromatography. A photometric assay revealed that His(6)-Tba solubilised in dodecylmaltoside possesses ATPase activity, characteristic for ABC-transporters.

The minimal replicon of the Streptomyces ghanaensis plasmid pSG5 identified by subcloning and Tn5 mutagenesis.

The cryptic plasmid pSG5 of Streptomyces ghanaensis 5/1B (DSM 2932) was characterized to have a molecular size of 12.7 kb and approximate copy number of 20-50 per chromosome. A bifunctional derivative, designated pSW344E, consisting of pSG5 and an Escherichia coli vector plasmid was constructed. Following Tn5 mutagenesis in E. coli, the replication functions of the mutagenized pSW344E plasmids were analysed in S.lividans. A 2 kb DNA fragment of the pSG5 replicon was found to carry replication functions. Subcloning of pSG5 DNA into various replication probe vectors resulted in the identification of the pSG5 minimal replicon, identical to the above mentioned 2 kb DNA region. Several small bifunctional plasmids, able to replicate in E. coli as well as in Streptomyces, were generated during subcloning. Some of these plasmids were found to be useful shuttle vectors.

Phenanthroline-Cu(II) cleavage as a probe of rRNA structure.

Chemical cleavage is developing into a powerful tool for analysis and characterization of nucleic acids. Phenanthroline-Cu(II) cleavage has been used extensively for studies of DNA for the last two decades, but recently has been applied to structural studies of RNA as well. This approach has been used to study the structure and structural changes occurring in ribosomal RNA within the ribosomes. In this article we discuss the mechanism by which phenanthroline cleaves, the applications possible using this approach, and the results that can be obtained. Protocols for use of phenanthroline are outlined as well.

The binding interaction of Coomassie blue with proteins.

The Coomassie brilliant blue protein assay is commonly used because of its sensitivity and convenience, but it is not well understood on a molecular level. This study attempts to gain better understanding of the assay system through spectrophotometric binding studies carried out on selected proteins under solvent conditions characteristic of the normal protein assay. The studies were generally conducted at high protein/dye concentration ratios, where only the high-affinity dye binding sites would be occupied. Modified Scatchard and Hill analyses show that these high-affinity sites are few in number compared to the total number of dye-binding sites on a given protein. The magnitudes of the high-affinity binding constants are typical of noncovalent binding interactions.

[Prognostic scores in peritonitis: the Mannheim Peritonitis Index or APACHE II?]. / Prognosescores bei Peritonitis: Mannheimer Peritonitis-Index oder APACHE II?

Scoring systems are mandatory to quantify the severity of abdominal sepsis on the basis of objective criteria. The Mannheim Peritonitis Index (MPI) is a disease-specific score based on easy to handle clinical parameters. APACHE II is a large-scale general scoring system with acute physiological and chronic health parameters. To evaluate the prognostic value of both systems 108 patients with severe abdominal infection managed by open treatment entered a prospective study. 32 patients (29.6%) died, 23 of them due to sepsis and 9 from other causes. Both MPI and APACHE II scores correlated closely with mortality, with three and two significantly different classes, respectively. Statistical validation showed a sensitivity of 93% and a specificity of 16% for MPI, and 89% and 25% for the APACHE II. ROC curves were nearly parallel for both scores. In conclusion there was no significant difference in prognostic value between the scoring systems.

Evidence that an additional mutation is required to tolerate insertional inactivation of the Streptomyces lividans recA gene.

In contrast to recA of other bacteria, the recA gene of Streptomyces lividans has been described as indispensable for viability (G. Muth, D. Frese, A. Kleber, and W. Wohlleben, Mol. Gen. Genet. 255:420-428, 1997.). Therefore, a closer analysis of this gene was performed to detect possible unique features distinguishing the Streptomyces RecA protein from the well-characterized Escherichia coli RecA protein. The S. lividans recA gene restored UV resistance and recombination activity of an E. coli recA mutant. Also, transcriptional regulation was similar to that of E. coli recA. Gel retardation experiments showed that S. lividans recA is also under control of the Streptomyces SOS repressor LexA. The S. lividans recA gene could be replaced only by simultaneously expressing a plasmid encoded recA copy. Surprisingly, the recA expression plasmid could subsequently be eliminated using an incompatible plasmid without the loss of viability. Besides being UV sensitive and recombination deficient, all the mutants were blocked in sporulation. Genetic complementation restored UV resistance and recombination activity but did not affect the sporulation defect. This indicated that all the recA mutants had suffered from an additional mutation, which might allow toleration of a recA deficiency.

Mutational analysis of the Streptomyces lividans recA gene suggests that only mutants with residual activity remain viable.

Temperature-sensitive integration plasmids carrying internal fragments of the Streptomyces lividans TK24 recA gene were constructed and used to inactivate the chromosomal recA gene of S. lividans by gene disruption and gene replacement. Integration of these plasmids resulted in recA mutants expressing C-terminally truncated RecA proteins, as deduced from Southern hybridization experiments. Mutants FRECD2 in which the last 42 amino acids, comprising the variable part of bacterial RecA proteins, had been deleted retained the wild-type phenotype. The S. lividans recA mutant FRECD3 produced a RecA protein lacking 87 amino acids probably including the interfilament contact site. FRECD3 was more sensitive to UV and MMS than the wild-type. Its ability to undergo homologous recombination was impaired, but not completely abolished. Integration of the disruption plasmid pFRECD3 in S. coelicolor "Müller" caused the same mutant phenotype as S. lividans FRECD3. In spite of many attempts no S. lividans recA mutants with deletions of 165 C-terminal amino acids or more were isolated. Furthermore, the recA gene could not be replaced by a kanamycin resistance cassette. These experiments indicate a crucial role of the recA gene in ensuring viability of Streptomyces.

Transcriptional and mutational analyses of the Streptomyces lividans recX gene and its interference with RecA activity.

The role of the 20,922-Da RecX protein and its interference with RecA activity were analyzed in Streptomyces lividans. The recX gene is located 220 bp downstream of recA. Transcriptional analysis by reverse transcriptase PCR demonstrated that recX and recA constitute an operon. While recA was transcribed at a basal level even under noninducing conditions, a recA-recX cotranscript was only detectable after induction of recA following DNA damage. The recA-recX cotranscript was less abundant than the recA transcript alone. The recX gene was inactivated by gene replacement. The resulting mutant had a clearly diminished colony size, but was not impaired in recombination activity, genetic instability, and resistance against UV irradiation. Expression of an extra copy of the S. lividans recA gene under control of the thiostrepton-inducible tipA promoter was lethal to the recX mutant, demonstrating that RecX is required to overcome the toxic effects of recA overexpression. Since inactivation of the recX gene did not influence transcription of recA, the putative function of the RecX protein might be the downregulation of RecA activity by interaction with the RecA protein or filament.

Using a targeted chemical nuclease to elucidate conformational changes in the E. coli 30S ribosomal subunit.

Determining the detailed tertiary structure of 16S rRNA within 30S ribosomal subunits remains a challenging problem. The particular structure of the RNA which allows tRNA to effectively interact with the associated mRNA during protein synthesis remains particularly ambiguous. This study utilizes a chemical nuclease, 1, 10-o-phenanthroline-copper, to localize regions of 16S rRNA proximal to the decoding region under conditions in which tRNA does not readily associate with the 30S subunit (inactive conformation), and under conditions which optimize tRNA binding (active conformation). By covalently attaching 1,10-phenanthroline-copper to a DNA oligomer complementary to nucleotides in the decoding region (1396-1403), we have determined that nucleotides 923-929, 1391-1396, and 1190-1192 are within approximately 15 A of the nucleotide base-paired to nucleotide 1403 in inactive subunits, but in active subunits only cleavages (1404-1405) immediately proximal to the 5' end of the hybridized probe remain. These results provide evidence for dynamic movement in the 30S ribosomal subunit, reported for the first time using a targeted chemical nuclease.

Streptomyces ghanaensis plasmid pSG5: nucleotide sequence analysis of the self-transmissible minimal replicon and characterization of the replication mode.

The naturally temperature-sensitive plasmid pSG5 of Streptomyces ghanaensis DSM 2932 is the basis replicon of the "pGM-vectors." The nucleotide sequence of the pSG5 minimal replicon was determined. Only one single open reading frame (rep) with high coding probability is located on the minimal replicon. The deduced Rep protein consists of 378 aa and contains motifs characteristic of initiation proteins for rolling-circle-type replication. Sequence similarity indicated that the Rep protein of pSG5 is related to the Rep proteins of the pC194 plasmid family. Accumulation of large amounts of single-stranded plasmid DNA was shown for all small pGM vectors. A minus origin for the lagging strand synthesis was localized outside of the pSG5 minimal replicon. Although the sequenced pSG5 fragment is self-transmissible, it seems not to carry further genes in addition to the rep gene. This suggests that the transfer mechanism of plasmid pSG5 differs from that of other Streptomyces plasmids, which all encode specific transfer genes.

Cleavage of a 23S rRNA pseudoknot by phenanthroline-Cu(II).

Studying the intricate folding of rRNA within the ribosome remains a complex problem. Phenanthroline-Cu(II) complexes cleave phosphodiester bonds in rRNA in specific regions, apparently especially where the rRNA structure is constrained in some fashion. We have introduced phenanthroline-copper complexes into 50S Escherichia coli ribosomal subunits and shown specific cleavages in the regions containing nucleotides 60-66 and 87-100. This specificity of cleavage is reduced when the ribosome is heated to 80 degrees C and reduced to background when the ribosomal proteins are extracted and the cleavage repeated on protein-free 23S rRNA. It has been suggested that nucleotides 60-66 and 87-95 in E.coli 23S rRNA are involved in a putative pseudoknot structure, which is supported by covariance data. The paired cleavages of nearly equal intensity of these two regions, when in the ribosome, may further support the existence of a pseudoknot structure in the 100 region of 23S rRNA.

Positions in the 30S ribosomal subunit proximal to the 790 loop as determined by phenanthroline cleavage.

Positioning rRNA within the ribosome remains a challenging problem. Such positioning is critical to understanding ribosome function, as various rRNA regions interact to form suitable binding sites for ligands, such as tRNA and mRNA. We have used phenanthroline, a chemical nuclease, as a proximity probe, to help elucidate the regions of rRNA that are near neighbors of the stem-loop structure centering at nt 790 in the 16S rRNA of the Escherichia coli 30S ribosomal subunit. Using phenanthroline covalently attached to a DNA oligomer complementary to nt 787-795, we found that nt 582-584, 693-694, 787-790, and 795-797 were cleaved robustly and must lie within about 15 A of the tethered site at the 5' end of the DNA oligomer, which is adjacent to nt 795 of 16S rRNA.