The opcA and ΨopcB regions in Neisseria: genes, pseudogenes, deletions, insertion elements and DNA islands

Previous data have indicated that the opc gene encoding an immunogenic invasin is specific to Neisseria meningitidis (Nm) and is lacking in Neisseria gonorrhoeae (Ng). The data presented here show that Nm and Ng both contain two paralogous opc-like genes, opcA, corresponding to the former opc gene, and (psi)opcB, a pseudogene. The predicted OpcA and OpcB proteins possess transmembrane regions with conserved non-polar faces but differ extensively in four of the five surface-exposed loops. Gonococcal OpcA was expressed weakly under in vitro conditions, and it is unknown whether these bacteria can express this protein at high levels. Analysis of the sequences flanking opcA and (psi)opcB revealed a framework of conserved housekeeping genes interspersed with DNA islands. These regions also contained several pseudogenes, deletions and IS elements, attesting to considerable genome plasticity. Both opcA and (psi)opcB are located on DNA islands that have probably been imported from unrelated bacteria. A third island encodes the dcmD/dcrD R/M genes in Ng versus a small open reading frame in most strains of Nm. Rare strains of Nm were identified in which the R/M island has been imported. DNA islands in Nm and Ng seem to have been acquired by recombination via conserved flanking housekeeping genes rather than by insertion of mobile genetic elements.     

The glucose transport system of the hyperthermophilic anaerobic bacterium Thermotoga neapolitana

The glucose transport system of the extremely thermophilic anaerobic bacterium Thermotoga neapolitana was studied with the nonmetabolizable glucose analog 2-deoxy-D-glucose (2-DOG). T. neapolitana accumulated 2-DOG against a concentration gradient in an intracellular free sugar pool that was exchangeable with external source of energy, such as pyruvate, and was inhibited by arsenate and gramicidin D. There was no phosphoenolpyruvate-dependent phosphorylation of glucose, 2-DOG, or fructose by cell extracts or toluene-treated cells, indicating the absence of a phosphoenolpyruvate:sugar phosphotransferase system. These data indicate that D-glucose is taken up by T. neapolitana via an active transport system that is energized by an ion gradient generated by ATP, derived from substrate-level phosphorylation.     

Fucosylated protein of retinal cone photoreceptor outer segments: morphological and biochemical analyses

Cone outer segments (OS) of the goldfish retina are diffusely labeled after intravitreal injection of [(3)H]fucose while rod OS remain unlabeled. By electron microscopic radioautography, the OS of red- and blue-sensitive cones are heavily labeled while green- sensitive cone OS are lightly labeled. The time-course and pattern of OS labeling in all cone types from 30 min to 24 h resemble that of incorporation of other sugars into rhodopsin in rod OS. The nature of the cone OS-specific fucosylated component(s) was examined using biochemical techniques. Cone OS were prelabeled by intravitreal injection of [(3)H]fucose 24 h before sacrifice. Photoreceptor OS were isolated using a discontinuous sucrose density gradient and it was verified by electron microscopic radioautography that the only source of radioactivity in the preparations was cone OS. The different cone types could be recognized by the heaviness of labeling, characteristic membrane spacing, and 'staining' of green cone OS in vitro with horseradish peroxidase. After acid hydrolysis of prelabeled photoreceptor membranes, 90 percent of the counts were in the neutral sugar fraction which was analyzed by thin-layer chromatography. Approximately 70 percent of the radioactivity co-chromatographed with authentic fucose. SDS-PAGE/fluorography of prelabeled photoreceptor membranes revealed a single radioactive component that was lightly stained with coomassie blue and showed an apparent molecular weight of 33,000. This cone-derived band was separated from unlabeled rod opsin which was well stained and showed an apparent mol wt of 38,000. Isoelectric focusing under denaturing conditions produced two major and one minor band of radioactivity with isoelectric points of 8.2, 8.6, and 8.8 respectively. No radioactivity was found in association with a stained band corresponding in isoelectric point to that of bovine opsin (pl, 6.2). The fucosylated component was readily digested by pronase, indicating its protein nature. Washing of the isolated OS with isotonic and hypotonic buffers failed to extract major amounts of the radioactivity, suggesting that the fucosylated component is an integral membrane protein. The presence of a fucosylated protein thus represents a major difference between cone and rod OS in the goldfish and has enabled us to identify cone OS in preparations of isolated photoreceptor membranes and to demonstrate the separation of a cone-derived glycoprotein from rod opsin.     

Promoter-distal region of the tra operon of F-like sex factor R100 in Escherichia coli K-12.

The distal region of the tra (transfer) operon of F-like plasmid R100 was investigated, using small plasmids derived from R100, primarily the plasmid pSM6. The transposon Tn5 (which confers kanamycin resistance) was inserted at different positions into pSM6, and the transposition derivatives were tested for ability to complement defined tra mutants of the F sex factor. Thus, the tra genes traH, G, T, and D were localized on the plasmid R100. A restriction map of pSM6 was constructed, and the locations of the insertions were mapped, using restriction endonuclease digestion of the plasmid DNA and exploiting the fact that several restriction sites are localized in the inverted repeat regions of the transposon. The gene products of the genes traG, S, T, and D were identified by radioactive labeling of proteins synthesized in minicells carrying the various insertion plasmids followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The presence of another transfer gene, traI, was inferred from these data. Another protein, the r2-A protein, was also identified, and its gene was mapped. On the basis of the data, a best-fit physical map of this region of the tra operon of R100 was constructed. The results confirmed that the general order and size of the distal transfer genes is as in the F sex factor, but showed that differences exist with respect to all of the gene products. The significance of these differences are discussed in the light of the genetic and physical homology (Manning et al., J. Bacteriol. 150:76-88) of the transfer regions.     

Sequence Diversity, Predicted Two-Dimensional Protein Structure, and Epitope Mapping of Neisserial Opa Proteins

The sequence diversity of 45 Opa outer membrane proteins from Neisseria meningitidis, Neisseria gonorrhoeae, Neisseria sicca, and Neisseria flava indicates that horizontal genetic exchange of opa alleles has been rare between these species. A two-dimensional structural model containing four surface-exposed loops was constructed based on rules derived from porin crystal structure and on conservation of sequence homology within transmembrane β-strands. The minimal continuous epitopes recognized by 23 monoclonal antibodies were mapped to loops 2 and 3. Some of these epitopes are localized on the bacterial cell surface, in support of the model.     

Cell-cell interactions in conjugating Escherichia coli: role of F pili and fate of mating aggregates.

An electron microscopic radioautographic study was made of tritiated thymidine incorporation into the genome of Escherichia coli PAT 84 and of tritiated meso-D,L-2,6-diaminopimelic acid (DAP) into the cell envelope. Pulse-labeled cells growing at 30 degrees C with a doubling time of 170 min were classified according to length by the method of agar filtration. Mathematical analysis of the length distribution led to the assumption of an exponential relation between length and time. A novel DNA replication pattern was found. Within the cell cycle DNA replication terminates at 70 min; then a gap follows of 64 min, after which DNA replication is initiated at 134 min. Thus, the C period is 106 min and the D period is 100 min. Cell constriction starts at 141 min and coincides with initiation of DNA replication. Detailed quantitative analysis of the [3H]thymidine grain frequency distribution allowed the distinction of three groups of cells. The first group incorporated no label, the second group an amount C, and the third group an amount 2 X C. The relative contribution of each group to a particular length class was determined. The data fitted very well into the DNA replication pattern. The same analysis was carried out on DAP pulse-labeled cells. Again, three groups of cells could be distinguished, and their relative contributions to each length class was determined. The group with the double amount of label was especially prominent at the end of the cell cycle. The emergence of this group might represent the acquisition of new lateral growth areas.     

Allelic polymorphism and site-specific recombination in the opc locus of Neisseria meningitidis

The opc gene is widespread in epidemic and endemic Neisseria meningitidis , but most strains of certain epidemic clones (ET-37 complex, Cluster A4) and a few random endemic isolates lack an opc gene. Four percent of the 1148 bp that contain opc plus the surrounding intergenic region was polymorphic (18 alleles), and many of the alleles contained a 230 bp insertion at a fixed location in the intergenic region. The presence or absence of the insertion reflects site-specific recombination. The alleles are stably inherited within clonal groupings for up to at least 50 years, with rare cases of horizontal genetic exchange. Most statistical methods indicated significant intragenic recombination events within this dataset.     

Identification of endogenous control genes for normalisation of real-time quantitative PCR data in colorectal cancer

Background  

Gene expression analysis has many applications in cancer diagnosis, prognosis and therapeutic care. Relative quantification is the most widely adopted approach whereby quantification of gene expression is normalised relative to an endogenously expressed control (EC) gene. Central to the reliable determination of gene expression is the choice of control gene. The purpose of this study was to evaluate a panel of candidate EC genes from which to identify the most stably expressed gene(s) to normalise RQ-PCR data derived from primary colorectal cancer tissue.     

Sex and virulence in Escherichia coli: an evolutionary perspective

Pathogenic Escherichia coli cause over 160 million cases of dysentery and one million deaths per year, whereas non-pathogenic E. coli constitute part of the normal intestinal flora of healthy mammals and birds. The evolutionary pathways underlying this dichotomy in bacterial lifestyle were investigated by multilocus sequence typing of a global collection of isolates. Specific pathogen types [enterohaemorrhagic E. coli, enteropathogenic E. coli, enteroinvasive E. coli, K1 and Shigella] have arisen independently and repeatedly in several lineages, whereas other lineages contain only few pathogens. Rates of evolution have accelerated in pathogenic lineages, culminating in highly virulent organisms whose genomic contents are altered frequently by increased rates of homologous recombination; thus, the evolution of virulence is linked to bacterial sex. This long-term pattern of evolution was observed in genes distributed throughout the genome, and thereby is the likely result of episodic selection for strains that can escape the host immune response.