Studies on Escherichia coli chromosome proteins. I. Analysis of the proteins by two-dimensional gel electrophoresis.

As an approach for studying the function of chromosome proteins in DNA replication and gene expression, proteins remaining attached to Escherichia coli nucleoids were analyzed by two-dimensional polyacrylamide slab gel electrophoresis. Nucleoids were isolated by gentle lysis of the cells in the presence of a DNA counter-ion such as 1 M NaCl or 5 mM spermidine. In exponentially growing cells, about 100 proteins have been found to exist in the nucleoids. Kinetic studies indicated that the number of chromosome proteins remaining attached varied with time after synchronization. Based on the pattern of the variation, appearance, increase or disappearance of the 29 major proteins, nucleoid proteins were shown to be classified in six different groups (groups A--F). A strong correlation was observed between the variation of proteins belonging to group D and initiation of DNA synthesis or cell division.

Restriction map of DNA spanning the replication terminus of the Bacillus subtilis chromosome.

The Bacillus subtilis 168 dna-1 chromosome was labelled during sporulation with [3H]thymine for five minutes immediately before termination of replication. The isolated radioactive DNA was cleaved with BamHI (or SalI) and the resulting restriction fragments separated by agarose gel electrophoresis. The individual fragments, fractionated into a series of slices cut from the gel, were then cleaved with SalI (or BamHI) and the double-digest fragments identified by electrophoresis and fluorography. All major fragments and most minor ones present in a whole double-digest were assigned to BamHI and SalI parents. Such information enabled the construction of an unambiguous restriction map of 150 X 10(3) bases of the approximately 250 X 10(3) bases of DNA labelled in the five minutes. In conjunction with published data on the order of replication of restriction fragments as termination is approached, it was clear that most (105 X 10(3) bases) of the mapped DNA was replicated by a major fork moving in one direction towards a BamHI 24.8 X 10(3) base fragment. The 45 X 10(3) bases extending to the other side of this region were labelled only slightly, and presumably was replicated by a fork that approached the other in an opposite direction until its progress was blocked or severely impeded within this region at a site, referred to as terC, sometime (less than 5 min) earlier. The regions of the map replicated in the final 2.5 and 1.0 minute by the major fork were also identified.

Mapping of Escherichia coli chromosomal Tn5 and F insertions by pulsed field gel electrophoresis.

A low resolution Not I physical map of Escherichia coli was recently constructed. In this report we demonstrated that this map can be used to map Tn5 and F insertions physically. The transposon, Tn5, contains Not I recognition sequences in its IS50 sequences. F plasmid contains an unmapped Not I site. Hence, the location of Tn5 and F in the chromosome can be mapped by identifying the location of the introduced Not I sites using pulsed field gel electrophoresis. The physical mapping of genetically mapped Tn5 insertions confirm the previously constructed Not I map and helps align the E. coli physical and genetic maps. The use of Tn5 can assist the construction of both physical and genetic maps for microorganisms lacking such maps. Variations on this approach will facilitate physical mapping with a wide variety of organisms, enzymes, and genetic elements.

Bacterial conjugation: an analysis of mixed recombinant clones.

A fraction of recombinant colonies resulting from conjugation is heterogenetic for unselected markers. Constitutivity for alkaline phosphatase synthesis (phoR) is studied as the unselected marker. The frequency of phoR heterogeneity depends on the genetic distance between phoR and the selected marker. Various models are considered which explain the formation of heterogenetic colonies (mixed clones), and experiments are described which test these models. It is concluded that the Hfr fragment can replicate and participate more than once in recombination thus yielding heterogenetic colonies.

Chromosome analysis by two-dimensional fingerprinting.

A two-dimensional fingerprinting technique has been developed that allows large, cell genome-size DNA's to be analyzed by restriction endonuclease cleavage and separation of DNA fragments by agarose gel electrophoresis. Equations have been derived to determine the genome size and number of cleavage sites from analysis of the distribution of fragment lengths. Genome sizes of Escherichia coli strain JM101 and two strains of the mycoplasma Acholeplasma laidlawii measured by this method are in agreement with published values. Other uses of two-dimensional fingerprinting for studies of prokaryotic and eukaryotic genome structure and organization are described.

A partial restriction map of the proA-purE region of the Escherichia coli K12 chromosome.

EcoRI restriction mapping data for fragments larger than 0.7 kb and contained in a 350-kb region of the Escherichia coli K-12 chromosome are presented. 75% of these fragments have been located relative to proA, B, argF, lac, proC, purE, and various insertion sequence elements normally present in this region. BglII and BamHI maps for the regions near argF and purE are also provided.

Comparison of IS1, IS2 and IS3 copy number in Escherichia coli strains K-12, B and C.

The number of copies of IS2 and IS1 in the chromosomes of five Escherichia coli K-12 strains and E. coli B and E. coli C has been determined by hybridization. Among these strains, IS1 copy numbers range from 4 to 19 and IS2 copy numbers range from 0 to 12. IS2 is present once in the E. coli B chromosome, but it is absent from E. coli C. The copy numbers of IS3 in the same seven E. coli strains range from 4 to 6.

Characterisation of methicillin-resistant Staphylococcus aureus isolates by restriction endonuclease digestion of chromosomal DNA.

Clinical isolates of Staphylococcus aureus from the London Hospital were characterised by genetic analysis of antibiotic-resistance determinants and by restriction endonuclease digestion of chromosomal DNA and compared with isolates from elsewhere in the UK. Restriction enzyme digestion of chromosomal DNA confirmed that a single strain of methicillin-resistant S. aureus (MRSA) persists at the London Hospital, although its antibiotic-resistance profile and plasmid carriage are not constant. Methicillin-sensitive isolates, on the other hand, each had readily distinguishable and unique DNA restriction patterns. The DNA restriction digest pattern of the London Hospital MRSA isolates was identical to that of "epidemic" (E) MRSA isolates from the Thames regions. By contrast, other MRSA isolates had DNA restriction patterns which differed from those of EMRSA isolates and from each other. These results confirm the discriminatory value of restriction pattern analysis as a typing method.

Characterization of chromosomal and membrane associated plasmid in Bacillus brevis ATCC 9999.

A covalently closed circular (ccc) DNA, with a weight of 44.7 x 10(6) daltons, has been isolated from Bacillus brevis ATCC 999 (a gramicidin S producer) and from the gramicidin S-negative mutant EB16. The ccc DNA in the case of the parent strain, is mainly (99%) attached to the chromosome and membrane fraction. A restriction enzyme map of the plasmid DNA was constructed for the enzymes SalI, SmaI and BamHI, which cleaved the plasmid DNA into two, two and six fragments respectively. Further digestion with the endonucleases EcoRI and HindIII cleaved the plasmid into 17 and 22 fragments.

[Structure of chromosomal deoxyribonucleoproteins. XI. Organization of deoxyribonucleoprotein complex in bacterial cells]. / Strukturakhromosomnykh dezoksiribonukleoproteidov. XI. Ob organizatsii dezoksiribonukleoproteidnogo kompleksa v bakterial'noi kletke.

Isolation of bacterial chromosomes under "mild" conditions enable us to purify a bacterial deoxyribonucleoprotein (DNP). This DNP is enriched in small basic proteins which are complexed with DNA throughout all the purification steps. The basic proteins are shown to be similar to some heat-resistant proteins of cellular extract and can interact with DNA in vitro. Extensive nuclease digestion of purified DNP of E. coli produced the smallest DNA fragments of 100--120 base pairs in length. The proteins of the DNP and the possible mode of arrangement of basic proteins along the DNA are discussed.

[Isolation and characteristics of plasmids determining multiple antibiotic resistance in strains of group A streptococci]. / Vydelenie i kharakteristika plazmidy, determiniruiushchei mnozhestvennuiu rezistentnost' k antibiotikam shtammov streptokokka gruppy A.

DNA of the streptococcal plasmid ERLI, determining resistance to erythromycin and lincomycin has been studied. The presence of satellite DNA component in streptococcal DNA has been demonstrated by dye-CsCl and CsCl density centrifugation, by chromatography of denatured-renatured DNA on the nitrocellulose and by electron microscopy. By all these methods the presence of covalently closed circular DNA molecules has been shown. Plasmid DNA has buoyant density in CsCl equal to 1.698 g/cm3 (GC content--38.8%), molecular weight (by electron microscopy) -- 19.8 Mdal; number of copies chromosomal genome equivalent is equal to about 4--4.5. Plasmid ERLI DNA was extracted from an original strain-carrier of plasmid ERLI, from a transduced strain which received plasmid ERLI as a result of transduction and lysogenization by phage "mo" and from the two antibiotic sensitive EMS mutants of resistant strain. Satellite DNA could not be isolated from a sensitive strain which served as a recipient in transduction experiments. The results obtained are in agreement with the literature on the molecular weight of the plasmid measured by the sedimentation analysis and with the previous genetic data on the antibiotic resistance within group A streptococci.

[Genetic approach to the virulence of bacteria]. / Approche génétique de la virulence des bactéries.

Bacteria only possess a single chromosome, but they also contain prophages, plasmids and transposons. The genetic basis for the pathogenic power of invasive and toxigenic bacteria only represents a small part of the total genotype of these bacteria. It is curious that although the plasmids and the prophages only represent about 1 p. cent of the total genome, they are disproportionately responsible for carrying the genetic information concerning virulence. The genetic study of virulence has revealed new mechanisms of pathogenic activity, for example the interaction of siderophores, adherence factors and haemolysins in pathogenic activity. Furthermore, the understanding of the genetic basis of virulence will lead to new methods of diagnosis and a new generation of vaccines.