Wolfgang Keck

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.

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 bacterial 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.

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.

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.

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 large protein. The homology between Slt35 and Slt70, albelt 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 microbial postgenomic era: promises, problems and prospects.

The inevitable emergence and spread of resistance to new antibiotics entering the market necessitates a new approach in drug discovery. Novel classes of antimicrobial compounds are required that are not enfeebled by widespread resistance mechanisms. The favoured approach is to gain a better understanding of the essential pathways and cellular functions and then to select new unexploited targets. This strategy has coincided with the deposition of fully assembled genomic sequences of several bacteria in the public databases. Various technologies have been reported that, when optimised, will enable the analysis of global cellular processes at the molecular level thus greatly contributing to our understanding of cellular physiology. In this article, some of the major advances in technology, which are expected in the future to be essential for the optimal use of the information contained within the genomic sequences, will be outlined.

Genome-wide transcriptional analysis of bacterial genomes: applications in antibacterial drug discovery

An oligonucleotide array of more than 250,000 specific probes representing two complete bacterial genomes including the 1,743 open reading frames of Haemophilus influenzae, and the 1,969 open reading frames of Streptococcus pneumoniae has been designed. When hybridised against labelled genomic DNA, all genes were detected and more than 96% of the signal intensity values were within a factor of three of the mean. For transcript imaging, microarrays were hybridised against total RNA populations quantitatively represented by labelled cDNAs. The measurements of transcript abundance for all genes were shown to be sensitive, specific, quantitative and reproducible. Nearly 85% of all S. pneumoniae mRNAs were found expressed during in vitro exponential growth. Identification of regulons important for S. pneumoniae entry into the stationary phase has been used to validate the approach. We will show, for the first time, how a genome-wide transcriptional analysis is applied to bacterial genomes and is being used to identify signal transduction regulatory networks, and to profile antibiotic compounds.