Identification of a putative chromosomal replication origin from Helicobacter pylori and its interaction with the initiator protein DnaA.

The key elements of the initiation of Helicobacter pylori chromosome replication, DnaA protein and putative oriC region, have been characterized. The gene arrangement in the H.pylori dnaA region differs from that found in many other eubacterial dnaA regions (rnpA-rmpH-dnaA-dnaN-recF-gyrB). Helicobacter pylori dnaA is flanked by two open reading frames with unknown function, while dnaN-gyrB and rnpA-rmpH loci are separated from the dnaA gene by 600 and 90 kb, respectively. We show that the dnaA gene encoding initiator protein DnaA is expressed in H.pylori cells. The H.pylori DnaA protein, like other DnaA proteins, can be divided into four domains. Here we demonstrate that the C-terminal domain of H.pylori DnaA protein is responsible for DNA binding. Using in silico and in vitro studies, the putative oriC region containing five DnaA boxes has been located upstream of the dnaA gene. DNase I and gel retardation analyses show that the C-terminal domain of H.pylori DnaA protein specifically binds each of five DnaA boxes.

Architecture of the Streptomyces lividans DnaA protein-replication origin complexes.

The Streptomyces oriC region contains two clusters of 19 DnaA boxes separated by a spacer (134 bp). The Streptomyces DnaA protein consists, like all other DnaA proteins, of four domains: domain III and the carboxyterminal part (domain IV) are responsible for binding of ATP and DNA, respectively. Binding of the DnaA protein to the entire oriC region analysed by electron microscopy showed that the DnaA protein forms separate complexes at each of the clusters of DnaA boxes, but not at the spacer separating them. In vivo mutational analysis revealed that the number of DnaA boxes and the presence of the spacer linking both groups of DnaA boxes seem to be important for a functional Streptomyces origin. We suggest that the arrangement of DnaA boxes allows the DNA-bound DnaA protein to induce bending and looping of the oriC region. As it was shown by electrophoretic mobility shift assay and "one hybrid system", two domains, I and III, facilitate interactions between DnaA molecules. We postulate that domain I and domain III could be involved in cooperativity at distant and at closely spaced DnaA boxes, respectively. The long domain II extends the range over which N termini (domain I) of DNA-bound DnaA protein can form dimers. Thus, interactions between DnaA molecules may bring two clusters of DnaA boxes separated by the spacer into functional contact by loop formation. Removal of the spacer region or deletion of domains I and II resulted, respectively, in nucleoprotein complexes which are not fully developed, or huge nucleoprotein aggregates.

Organization and nucleotide sequence analysis of the ribosomal gene set (rrnB) from Streptomyces lividans.

Streptomyces lividans (Sl) contains six ribosomal RNA (rRNA) gene sets, rrnA-F (Suzuki, Y., Ono, Y., Nagata, A. and Yamada, T. (1988) Molecular cloning and characterization of an rRNA operon in Streptomyces lividans TK21. J. Bacteriol. 170, 1631-1636). We have cloned the rrnB gene cluster. Physical mapping revealed that rrnB gene set is located on a 290 kb Asel fragment in the 11 to 12 o'clock region of the S. coelicolor A3(2) chromosome. The complete nucleotide (nt) sequence of Sl 23S rRNA has been determined. The structural gene of the Sl 23S rRNA codes for the 3108 nt RNA chain. The G+C content of the 23S rRNA is 57.3 mol%. The length of the spacer region between the 23S and 5S genes is 99 bp. Analysis of the sequences between the 16S and 23S genes and downstream of the 5S rRNA gene failed to identify any tRNA-like sequences. A secondary structure model of Sl 23 rRNA is proposed, based on the earlier published model of Gutell and Fox (Nucleic Acids Res. 16 (1988) 175-269).

Bacterial replication initiator DnaA. Rules for DnaA binding and roles of DnaA in origin unwinding and helicase loading.

We review the processes leading to the structural modifications required for the initiation of replication in Escherichia coli, the conversion of the initial complex to the open complex, loading of helicase, and the assembly of two replication forks. Rules for the binding of DnaA to its binding sites are derived, and the properties of ATP-DnaA are described. We provide new data on cooperative interaction and dimerization of DnaA proteins of E. coli, Streptomyces and Thermus thermophilus, and on the stoichiometry of DnaA-oriC complexes of E. coli.

Functional domains of DnaA proteins.

Functional domains of the initiator protein DnaA of Escherichia coli have been defined. Domain 1, amino acids 1-86, is involved in oligomerization and in interaction with DnaB. Domain 2, aa 87-134, constitutes a flexible loop. Domain 3, aa 135-373, contains the binding site for ATP or ADP, the ATPase function, a second interaction site with DnaB, and is required for local DNA unwinding. Domain 4 is required and sufficient for specific binding to DNA. We show that there are three different types of cooperative interactions during the DNA binding of DnaA proteins from E. coli, Streptomyces lividans, and Thermus thermophilus: i) binding to distant binding sites; ii) binding to closely spaced binding sites; and iii) binding to non-canonical binding sites.

pS10147-2, a 3.7 kb multicopy plasmid isolated from Streptomyces coelicolor.

The multicopy plasmid, pS10147-2 (3.7 kb) was isolated from Streptomyces coelicolor IMET 40271. A restriction enzyme map of pS10147-2 was constructed. The pS10147-2 is probably a spontaneous deletion derivative of pIJ101.

Identification of Staphylococcus epidermidis using a 16S rRNA-directed oligonucleotide probe.

Staphylococcus epidermidis is the most medically significant of the coagulase-negative staphylococci. An oligonucleotide probe (pSe) for identification of S. epidermidis was defined by comparing the sequences of the 16S rRNA variable region V6 from numerous coagulase-negative staphylococci. In order to increase the sensitivity of the detection, polymerase chain reaction amplification of the variable region with primers based on the conserved flanking sequences was applied. The detection limit of the polymerase chain reaction assay combined with pSe probe was shown to be 1 fg which corresponds to about one single bacterium. Additionally, a sensitive, non-radioisotopic system with chemiluminescence detection was tested.

Rare, suppurative pulmonary infection caused by Nocardiopsis dassonvillei recognized by glycolipid markers.

An opportunistic actinomycete was isolated as the only etiological agent of a severe, suppurative pulmonary infection. The strain was rapidly recognised as Nocardiopsis by the taxonomically important and immunologically active glycolipid markers (G1 and G2). Identification of the clinical isolate, from a group of actinomycetes mainly known as soil habitants, was definitely proved by chemotaxonomic studies (cell wall/sugar, phospholipid and fatty acid types) as well as by genomic data (GC content, DNA-DNA reassociation). The level of DNA-DNA homology of the clinical actinomycete, in comparison with other reference members of this genus, revealed the highest (88%) relatedness to Nocardiopsis dassonvillei. The results confirmed the value and generic specificity of glycolipid markers from Nocardiopsis, the first time used for rapid recognition of a clinical strain causing a nocardiosis-like disease.

Kitasatosporia setae, an interesting actinomycete strain isolated from Spitsbergen soil.

The phenotypic and genotypic properties as well as cell chemistry of an interesting actinomycete, which converted certain azacarbazoles into highly cytotoxic derivatives, were established. The strain was also compared with two similar Japanese actinomycetes of Kitasatosporia gen. nov. A great resemblance between the strains was observed. Apart from similar phenotypic properties they were characterized by contents of both L- and meso-DAP, glycine and galactose as the main cell wall components. They proved to have the same phospholipid, glycolipid and fatty acid patterns. The guanine-plus-cytosine contents of the deoxyribonucleic acids of the strains averaged 72.5 and there was a high degree of homology between the DNAs of the strains (approximately 80%). These data provide evidence that the Spitsbergen isolate and the Japanese actinomycetes belong to one genomic species.

DNA restriction endonuclease analysis of some Streptomyces species.

Relationship between some Streptomyces species were determined by restriction endonuclease analysis (REA). DNA isolated from twelve representative Streptomyces strains was analysed using Sal I endonuclease. The results indicate that REA patterns can be used to determine variation both between as well as within species of Streptomyces.

Rapid detection of Staphylococcus saprophyticus using primer specific PCR.

Staphylococcus saprophyticus is one of the most frequently encountered clinically significant members of the coagulase-negative staphylococci. A set of species-specific PCR primers was defined for the detection of Staphylococcus saprophyticus. These primers target variable regions (V3 and V6) of the 16S rRNA gene. Primer-specific PCR has potential applications in epidemiological studies and diagnosis of Staphylococcus saprophyticus.

[Initiation of DNA replication in Streptomyces; organizational comparison of the oriC region in prokaryotic organisms]. / Inicjacja replikacji DNA u Streptomyces; porównanie organizacji regionu oriC organizmów prokariotycznych.

DnaA protein and its binding sequence (DnaA-box) are two elements essential for the initiation of chromosomal replication in Escherichia coli and other bacteria. Recently these two elements have been found to be conserved in four Gram-positive bacteria (Bacillus subtilis, Micrococcus luteus, Mycoplasma capricolum and Streptomyces lividans). The structures of eubacterial DnaA-box regions and their locations on the chromosome and than functional aspects od DnaA protein have been compared.

[Ribosomal RNA-specific DNA probes used in microbiology]. / Sondy DNA komplementarne do rRNA--ich zastosowanie w mikrobiologii.

Recent advances in molecular biology and gene technology made it possible to identify microorganisms on the basis of most stable characters directly linked to the molecular structure of the genome. One of the most basic technics in this field is reassociation between two complementary strands of nucleic acids (DNA and RNA) isolates from different microorganisms. A probe is a specific fragment of single stranded nucleic acid (DNA or RNA) that binds to the complementary, target nucleic acid. Several aspects of probes designation and diagnostic application are presented and discussed.

Characterization of an autonomously replicating region from the Streptomyces lividans chromosome.

The chromosomal replication origin of the plasmidless derivative (TK21) from Streptomyces lividans 66 has been cloned as an autonomously replicating minichromosome (pSOR1) by using the thiostrepton resistance gene as a selectable marker. pSOR1 could be recovered as a closed circular plasmid which shows high segregational instability. pSOR1 was shown to replicate in Streptomyces coelicolor A3(2) and in S. lividans 66 and hybridized with DNA from several different Streptomyces strains. Physical mapping revealed that oriC is located on a 330-kb AseI fragment of the S. coelicolor A3(2) chromosome. DNA sequence analyses showed that the cloned chromosomal oriC region contains numerous DnaA boxes which are arranged in two clusters. The preferred sequence identified in the oriC region of Escherichia coli and several other bacteria is TTATCCACA. In contrast, in S. lividans, which has a high GC content, the preferred sequence for DnaA boxes appears to be TTGTCCACA.

[Organization of the Helicobacter pylori genome]. / Organizacja genomu Helicobacter pylori.

Helicobacter pylori is a Gram-negative, spiral-shaped pathogenic bacterium that was firstly isolated and cultured from biopsy specimens by Marshall and Warren in 1983. This organism is a human gastric pathogen associated with peptic ulcer disease as well as chronic gastritis. Recent epidemiological studies have demonstrated that H. pylori is a primary risk factor for the development of intestinal type gastric adenocarcinoma. H. pylori is the first bacterium for which the genomes of two unrelated strains (26695 and J99) have been sequenced. The genome of H. pylori is relatively low in size (1.6-1.73 Mb). In this review, we compare the organization of two sequenced H. pylori genomes. A special emphasis on genetic diversity of H. pylori including plasticity zone and cag pathogenicity island has been placed.

[Cytotoxin vacuolization of Helicobacter pylori]. / Cytotoksyna wakuolizujaca Helicobacter pylori.

Helicobacter pylori is now recognized as the major causative agent of chronic superficial gastritis in humans. The virulence factors of H. pylori are still poorly understood. Vacuolating cytotoxin (VacA) is one of the factors that has been identified so far. VacA induces cytoplasmic vacuolation in eukaryotic cells. The vacA gene encodes a precursor protein of 140 kDa which consists of a 33-amino acid signal sequence, the 87 kDa cytotoxin and a 50 kDa C-terminal domain. The 50 kDa domain is involved in translocation of VacA across outer membrane. Sequence analysis of vacA gene derived from different strains of H. pylori revealed the existence of several families of vacA gene allels. Analysis of a clinically isolated strains of H. pylori showed the correlation between presence of specific vacA allels, VacA activity and peptic ulceration.

DNA base composition and homology values in the classification of some Rhodococcus species.

Similar DNA homology values were recorded when a modified S1 nuclease technique and a standard nitrocellulose membrane filter method were applied to representative strains of Rhodococcus. The DNA homology data showed that R. globerulus, R. luteus and R. sputi form distinct genomic species. The congruence between the DNA homology and earlier numerical phenetic data was good, but there was evidence that some strains had been misclassified in the previous studies. In particular, the type strains of R. obuensis and R. sputi belong to a single genomic species. The former name is thus a later, subjective synonym of the latter. The guanine plus cytosine content of the DNA of the rhodococci fell within the range 61 to 72 mol%.

Deoxyribonucleic acid relatedness amongst Staphylococcus epidermidis and Staphylococcus saprophyticus strains.

The degree of binding was determined between DNA preparations from 65 staphylococci representing cluster defined in a numerical phenetic survey and 3H-labelled DNA from reference strains of S. epidermidis, S. haemolyticus, S. hominis, S. saprophyticus and S. warneri. The congruence between the DNA pairing and numerical phenetic data was good with S. epidermidis and S. saprophyticus being shown to be genomic species. However, some strains identified as S. epidermidis using recommended diagnostic procedures were found to belong to other taxa, notably S. simulans and S. warneri. The moles percent guanine plus cytosine content of the DNA of the test strains was within the range 27 to 34.