Bacterial transcript imaging by hybridization of total RNA to oligonucleotide arrays.

We have used high-density oligonucleotide probe arrays (chips) for bacterial transcript imaging. We designed a chip containing probes representing 106 Hemophilus influenzae genes and 100 Streptococcus pneumoniae genes. The apparent lack of polyadenylated transcripts excludes enrichment of mRNA by affinity purification and we thus used total, chemically biotinylated RNA as hybridization probe. We show that hybridization of Streptococcus RNA to a chip allows simultaneous quantification of the transcript levels. The sensitivity was found to be in the range of one to five transcripts per cell. The quantitative chip results were in good agreement with conventional Northern blot analysis of selected genes. This technology allows simultaneous and quantitative measurement of the transcriptional activity of entire bacterial genomes on a single oligonucleotide probe array.

Crystal structure of Escherichia coli lytic transglycosylase Slt35 reveals a lysozyme-like catalytic domain with an EF-hand.

BACKGROUND: Lytic transglycosylases are bacterial muramidases that catalyse the cleavage of the beta- 1,4-glycosidic bond between N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) in peptidoglycan with concomitant formation of a 1,6-anhydrobond in the MurNAc residue. These muramidases play an important role in the metabolism of the bacterial cell wall and might therefore be potential targets for the rational design of antibacterial drugs. One of the lytic transglycosylases is Slt35, a naturally occurring soluble fragment of the outer membrane bound lytic transglycosylase B (MltB) from Escherichia coli. RESULTS: The crystal structure of Slt35 has been determined at 1.7 A resolution. The structure reveals an ellipsoid molecule with three domains called the alpha, beta and core domains. The core domain is sandwiched between the alpha and beta domains. Its fold resembles that of lysozyme, but it contains a single metal ion binding site in a helix-loop-helix module that is surprisingly similar to the eukaryotic EF-hand calcium-binding fold. Interestingly, the Slt35 EF-hand loop consists of 15 residues instead of the usual 12 residues. The only other prokaryotic proteins with an EF-hand motif identified so far are the D-galactose-binding proteins. Residues from the alpha and core domains form a deep groove where the substrate fragment GlcNAc can be bound. CONCLUSIONS: The three-domain structure of Slt35 is completely different from the Slt70 structure, the only other lytic transglycosylase of known structure. Nevertheless, the core domain of Slt35 closely resembles the fold of the catalytic domain of Slt70, despite the absence of any obvious sequence similarity. Residue Glu162 of Slt35 is in an equivalent position to Glu478, the catalytic acid/base of Slt70. GlcNAc binds close to Glu162 in the deep groove. Moreover, mutation of Glu162 into a glutamine residue yielded a completely inactive enzyme. These observations indicate the location of the active site and strongly support a catalytic role for Glu162.

Purification and characterization of N-acetylmuramyl-L-alanine amidase from human plasma using monoclonal antibodies.

N-Acetylmuramyl-L-alanine amidase (EC 3.5.1.28) cleaves the amide bond between N-acetyl muramic acid and L-alanine in the peptide side chain of different peptidoglycan products. The enzyme was purified from human plasma using a three-step column chromatography procedure. Monoclonal antibodies were produced against the purified human enzyme. By coupling of a high affinity monoclonal antibody to sepharose beads an immunoadsorbent column was prepared. Using this second purification method it was possible to purify large amounts of the amidase from human plasma in a single step. SDS-PAGE showed one single band of 70 kDa and two-dimensional electrophoresis showed the presence of multiple isomeric forms of the protein with pI between 6.5 and 7.9. Two different methods were used for determination of substrate specificity, a HPLC method separating peptidoglycan monomers from the reaction products after incubation with amidase and a colorimetric method when high molecular weight peptidoglycan was used as a substrate for amidase. It is shown that the disaccharide tetra peptide, disaccharide penta peptide and the anhydro disaccharide tetrapeptide are good substrates for the amidase and that muramyl dipeptide and disaccharide dipeptide are not a substrate for the amidase. Using one of the monoclonal antibodies against the amidase it was shown in FACScan analysis that N-acetylmuramyl-L-alanine amidase is present in granulocytes but not in monocytes from unstimulated peripheral blood of a healthy donor. The presence of N-acetylmuramyl-L-alanine amidase in granulocytes is a novel finding and perhaps important for the inactivation of biologically active peptidoglycan products still present after hydrolysis by lysozyme.

Crystallization of the soluble lytic transglycosylase from Escherichia coli K12.

Lytic transglycosylases degrade the murein polymer of the bacterial cell wall to 1,6-anhydromuropeptides. These enzymes are of significant medical interest, not only because they are ideal targets for the development of new classes of antibiotics, but also because the low molecular weight products of their catalytic action can cause diverse biological activities in humans, which can be either beneficial or toxic. A soluble lytic transglycosylase was purified from an overproducing Escherichia coli strain and X-ray quality crystals were obtained at room temperature from hanging drops by vapor diffusion against 20 to 25% (NH4)2SO4, in 100 mM-sodium acetate buffer, pH 5.0. The crystals diffract in the X-ray beam to 2.8 A resolution. Their space group is P2(1)2(1)2(1) with cell dimensions a = 81 A, b = 88 A and c = 135 A. Assuming one monomer (Mr 70,362) per asymmetric unit, the solvent content of these crystals is 63%.

Crystallization of a genetically engineered water-soluble primary penicillin target enzyme. The high molecular mass PBP2x of Streptococcus pneumoniae.

A genetically engineered water-soluble derivative of PBP2x of Streptococcus pneumoniae has been produced, purified and crystallized in a form suitable for X-ray diffraction analysis. The best crystals have been grown at 15 degrees C, from solutions containing 8% polyethylene glycol 10,000 at pH values ranging from 3.9 to 6.0. These crystals diffract to a resolution of 3.5 A and have a space group P6(1)22 (or enantiomorph) with unit cell dimensions of a = b = 162.2 A, c = 171.8 A, alpha = beta = 90 degrees, gamma = 120 degrees. The molecular mass and cell dimensions suggest that there is one molecule of enzyme per asymmetric unit. The breakdown of a chromogenic cephalosporin derivative diffused into a crystal reveals clearly that the enzyme is active in the crystalline state.

Evaluation of a fall detector based on accelerometers: a pilot study.

As falls and fall-related injuries remain a major challenge in the public health domain, reliable and immediate detection of falls is important so that adequate medical support can be delivered. Available home alarm systems are placed on the hip, but have several shortcomings. A fall detector based on accelerometers and placed at head level was developed, as well as an algorithm able to distinguish between activities of daily living and simulated falls. Accelerometers were integrated into a hearing-aid housing, which was fixed behind the ear. The sensitivity of the fall detection was assessed by investigation into the acceleration patterns of the head of a young volunteer during intentional falls. The specificity was assessed by investigation into activities of daily living of the same volunteer. In addition, a healthy elderly woman (83 years) wore the sensor during the day. Three trigger thresholds were identified so that a fall could be recognised: the sum-vector of acceleration in the xy-plane higher than 2 g; the sum-vector of velocity of all spatial components right before the impact higher than 0.7 m s(-1); and the sum-vector of acceleration of all spatial components higher than 6 g. The algorithm was able to discriminate activities of daily living from intentional falls. Thus high sensitivity and specificity of the algorithm could be demonstrated that was better than in other fall detectors worn at the hip or wrist at the same stage of development.

Integrational plasmids for the tetracycline-regulated expression of genes in Streptococcus pneumoniae.

Plasmids for the tetracycline regulated gene expression in Streptococcus pneumoniae have been developed. The plasmids were used for the tetracycline-dependent production of firefly luciferase in streptococci. The production of luciferase can be induced fivefold by the addition of tetracycline. By using two promoters of different strength and depending on the presence or absence of tetracycline, an 80-fold range of luciferase activities can be covered. The system was also used to construct strains that depend on the addition of tetracycline for the production of the A subunit of DNA gyrase, an essential streptococcal protein. The growth of such a strain depends on the addition of tetracycline to the medium. In the absence of tetracycline, the cells cease to grow and are not viable. The system presented in this report should be useful for the characterization of gene networks in S. pneumoniae. It especially allows one to study the function of essential genes that can not be investigated by standard knock-out techniques.

Domain organization and molecular characterization of 13 two-component systems identified by genome sequencing of Streptococcus pneumoniae.

In bacteria, adaptive responses to environmental stimuli are often initiated by two-component signal transduction systems (TCS). The prototypical TCS comprises two proteins: a histidine kinase (HK, hk) and a response regulator (RR rr). Recent research has suggested that compounds that inhibit two-component systems might have good antibacterial activity. In order to identify TCS that are crucial for growth or virulence of Streptococcus pneumoniae, we have examined the genomic sequence of a virulent S. pneumoniae strain for genes that are related to known histidine kinases or response regulators. Altogether 13 histidine kinases and 13 response regulators have been identified. The protein sequences encoded by these genes were compared with sequences deposited in public databases. This analysis revealed that two of the 13 pneumococcal TCSs have been described before (ciaRH and comDE) and two are homologous to the yycFG and the phoRP genes of Bacillus subtilis. All the pneumococcal response regulators contain putative DNA binding motifs within the C-terminal output domain, implying that they are involved in transcriptional control. Two of these response regulators are obviously the first representatives of a new subfamily containing an AraC-type DNA-binding effector domain. To assess the regulatory role of these transcription factors, we disrupted each of the 13 response regulator genes by insertional mutagenesis. All the viable mutant strains with disrupted response regulator genes were further characterized with regard to growth in vitro, competence, and experimental virulence. Two response regulator genes could not be inactivated, indicating that they may regulate essential cellular functions. The possibility of using these systems as targets for the development of novel antibacterials will be discussed.

Cloning and controlled overexpression of the gene encoding the 35 kDa soluble lytic transglycosylase from Escherichia coli.

The lytic transglycosylases of Escherichia coli are involved in peptidoglycan metabolism and resemble the lysozymes not only in activity, but in the case of the 70 kDa soluble lytic transglycosylase (Slt70), also structurally. Here we report the cloning of the gene that encodes the 35 kDa soluble lytic transglycosylase (Slt35) of E. coli. Based on the sequence of the full-length gene, Slt35 is very likely to be a proteolytically truncated form of a slightly larger protein. The homology between Slt35 and Slt70, albeit poor, indicates that the active site architecture of both proteins may be alike. Using the T-7 promoter system, Slt35 was overproduced in large quantities and purified to homogeneity for crystallographic purposes.

The monofunctional glycosyltransferase of Escherichia coli is a member of a new class of peptidoglycan-synthesising enzymes.

Using conserved fingerprints in the glycosyltransferase (GTase) domain of high-molecular-weight penicillin-binding proteins (PBP), a gene (mgt) encoding a putative monofunctional glycosyltransferase has been identified in Haemophilus influenzae and in other bacteria] species. Here we report the cloning of the homologous Escherichia coli gene and show that the solubilised membrane fraction of E. coli cells overexpressing the mgt gene contain a significantly increased peptidoglycan synthesis activity. In contrast to the high-molecular-weight PBPs, this activity is not inhibited by Flavomycin.

Identification of new members of the lytic transglycosylase family in Haemophilus influenzae and Escherichia coli.

Although bacterial peptidoglycan metabolism and numerous of the enzymes involved therein have been studied extensively over the years, information on the precise number of these enzymes is still lacking as is knowledge on the specific function of most of them. This observation holds true even for the well-studied bacterium Escherichia coli. Through determination of the complete sequences of bacterial genomes, that of Haemophilus influenzae being the first example, the opportunity arises to obtain a comprehensive overview of the members of the different families of peptidoglycan metabolizing enzymes by identification of their genes. Following this rationale, H. influenzae and E. coli genomic sequence was searched for new members of the family of lytic transglycosylases, using three-dimensional structure-derived sequence information. A new putative lytic transglycosylase gene could be identified in both bacterial species. The gene from E. coli was cloned and peptidoglycan hydrolase activity was demonstrated for the gene product.

Penicillin-binding protein 4 of Escherichia coli shows a novel type of primary structure among penicillin-interacting proteins.

The nucleotide sequence of a 1884 bp DNA fragment of E. coli, carrying the gene dacB, was determined. The DNA codes for penicillin-binding protein 4 (PBP4), an enzyme of 477 amino acids, being involved as a DD-carboxypeptidase-endopeptidase in murein metabolism. The enzyme is translated with a cleavable signal peptide of 20 amino acids, which was verified by sequencing the amino-terminus of the isolated protein. The characteristic active-site fingerprints SXXK, SXN and KTG of class A beta-lactamases and penicillin-binding proteins were located in the sequence. On the basis of amino acid alignments we propose, that PBP4 and class A beta-lactamases share a common evolutionary origin but PBP4 has acquired an additional domain of 188 amino acids in the region between the SXXK and SXN elements.

Control of the activity of the soluble lytic transglycosylase by the stringent response in Escherichia coli.

The soluble lytic transglycosylase (Slt) of Escherichia coli is known to be a powerful murein hydrolase in vitro. It is shown here to act as an autolysin in vivo as well. Rapid autolysis of Slt overproducing cells was induced by protein biosynthesis inhibitors, which also block the fomration of guanosine-5'-diphosphate-3'-diphosphate (ppGpp). When amino acid starvation was used to inhibit protein synthesis, autolysis was suppressed in relA+ but not in relA- cells. These findings indicate that the stringent control modulates the enzymatic activity of the soluble lytic transglycosylase in vivo.

In vivo crystal formation in Escherichia coli of an over-expressed soluble form of penicillin-binding protein 5.

Accumulation of either native membrane-bound or soluble variants of PBP5 over-expressed in the cytoplasm was investigated by electron microscopy of ultra-thin sections. One of the soluble forms of PBP5 (PBP5s353) formed well-ordered crystals inside the cells. Cells sectioned perpendicular to their long axis showed a diamond-shaped crystal whereas cells cut parallel to their long axis contained a long, narrow crystal. In both sectioning directions an ordered ultrastructure was visible as shown by optical diffraction. Computer processing was used to enhance the crystal images. From this the unit cell parameters were calculated as a = 7.6 nm, b = 4 nm, c = 4.2 nm, gamma = 75 degrees. The calculated unit-cell volume of 120 nm3 is large enough to contain one protein molecule.

[Volumetric and morphological CT parameters for assessing prognosis and treatment control in hepatocellular carcinoma undergoing arterial chemoembolization]. / Volumetrische und morphologische CT-Parameter zur Prognoseabschätzung und Therapiekontrolle des hepatozellulären Karzinoms unter arterieller Chemoembolisation.

PURPOSE: To evaluate volumetric CT data as a guide for indication and assessment of prognosis for transarterial chemo-embolisation (TAE) of hepatocellular carcinomas (HCC). MATERIAL AND METHOD: 74 patients with HCC were treated with repeated TAE. 50 mg Adriblastin/m2 body surface, 50 mg cisplatin/m2 body surface and 10 ml lipiodol were combined with 2-10 ml Spherex and injected selectively (25 cases) or superselectively (49 cases); in 28 patients a single injection and in 46 patients multiple injections were used. RESULTS: CT findings before and after the procedure showed a solitary lesion in 17 patients, two lesions in 18 patients and in 39 patients there were three or more lesions. Mean expectation of life was 523 days (median = 372 days; 57% of one year survival probability). In 29 patients with > 75% lipiodol retention, mean survival was 819 days; in 17 patients with < 75% lipiodol retention it was 382 days; lower lipiodol retention of < 50% it was 231 days (< 25% 192 and < 10% 152 days). A statistically significant relationship (p < 0.0001) could be established between survival time and tumour volume, relationship of tumour to liver volume, intratumoral lipiodol retention, the type of tumour growth and the number of liver segments involved. CONCLUSION: TAE provides best survival rates after repeated injections of solitary HCC with tumour volumes < 50 ml and > 75% intra-tumoral lipiodol retention.

[Agreement of automatically and manually determined parameters of the electronystagmogram]. / Untersuchung der Ubereinstimmung automatisch und manuell ermittelter Parameter des Elektronystagmogramms.

Prerequisite for clinical use of programs for automatic analysis of nystagmus is a similar accuracy in determining parameters of electronystagmograms in comparison with manual evaluation. For examination of accuracy and reliability of our program NYSLYS, we compared maximal values of the slow phase velocity (SPV) and the number of beats in 10 s of responses from thermal tests, with 100 patients with equilibrium disturbances. The average difference between the automatically and manually evaluated values for SPV and the number of beats in 10 s is in the range of +/- 15%. Correlation coefficients are 0.97 for SPV and 0.95 for the number of beats in 10 s. Similar correlation coefficients are obtained for the ratio of responses after left and right ear stimulation. These results show that our automatic analysis can replace tedious manual evaluation in most cases. In addition the temporal course of SPV is obtained as a good aid in the judgement of nystagmus. Automatic analysis, however, cannot fully replace the ability to examine electronystagmograms. Especially if curves are disturbed by many artifacts or in cases with very irregular reactions the original electronystagmogram must be considered, too.