The genome of Salinibacter ruber: convergence and gene exchange among hyperhalophilic bacteria and archaea.

Saturated thalassic brines are among the most physically demanding habitats on Earth: few microbes survive in them. Salinibacter ruber is among these organisms and has been found repeatedly in significant numbers in climax saltern crystallizer communities. The phenotype of this bacterium is remarkably similar to that of the hyperhalophilic Archaea (Haloarchaea). The genome sequence suggests that this resemblance has arisen through convergence at the physiological level (different genes producing similar overall phenotype) and the molecular level (independent mutations yielding similar sequences or structures). Several genes and gene clusters also derive by lateral transfer from (or may have been laterally transferred to) haloarchaea. S. ruber encodes four rhodopsins. One resembles bacterial proteorhodopsins and three are of the haloarchaeal type, previously uncharacterized in a bacterial genome. The impact of these modular adaptive elements on the cell biology and ecology of S. ruber is substantial, affecting salt adaptation, bioenergetics, and photobiology.

Radical-induced oxidation of metformin.

Metformin (1,1-dimethylbiguanide) is an antihyperglycaemic drug used to normalize glucose concentrations in type 2 diabetes. Furthermore, antioxidant benefits have been reported in diabetic patients treated with metformin. This work was aimed at studying the scavenging capacity of this drug against reactive oxygen species (ROS) like *OH and (O2*-)-free radicals. ROS were produced by gamma radiolysis of water. The irradiated solutions of metformin were analyzed by UV/visible absorption spectrophotometry. It has been shown that hydroxyl free radicals react with metformin in a concentration-dependent way. The maximum scavenging activity was obtained for concentrations of metformin > or = 200 micromol.L(-1), under our experimental conditions. An estimated value of 10(7) L.mol(-1).s(-1) has been determined for the second order rate constant k(*OH + metformin). Superoxide free radicals and hydrogen peroxide do not initiate any oxidation on metformin in our in vitro experiments.

Genome of Geobacter sulfurreducens: metal reduction in subsurface environments.

The complete genome sequence of Geobacter sulfurreducens, a delta-proteobacterium, reveals unsuspected capabilities, including evidence of aerobic metabolism, one-carbon and complex carbon metabolism, motility, and chemotactic behavior. These characteristics, coupled with the possession of many two-component sensors and many c-type cytochromes, reveal an ability to create alternative, redundant, electron transport networks and offer insights into the process of metal ion reduction in subsurface environments. As well as playing roles in the global cycling of metals and carbon, this organism clearly has the potential for use in bioremediation of radioactive metals and in the generation of electricity.

Genome sequence of Chlamydophila caviae (Chlamydia psittaci GPIC): examining the role of niche-specific genes in the evolution of the Chlamydiaceae.

The genome of Chlamydophila caviae (formerly Chlamydia psittaci, GPIC isolate) (1 173 390 nt with a plasmid of 7966 nt) was determined, representing the fourth species with a complete genome sequence from the Chlamydiaceae family of obligate intracellular bacterial pathogens. Of 1009 annotated genes, 798 were conserved in all three other completed Chlamydiaceae genomes. The C.caviae genome contains 68 genes that lack orthologs in any other completed chlamydial genomes, including tryptophan and thiamine biosynthesis determinants and a ribose-phosphate pyrophosphokinase, the product of the prsA gene. Notable amongst these was a novel member of the virulence-associated invasin/intimin family (IIF) of Gram-negative bacteria. Intriguingly, two authentic frameshift mutations in the ORF indicate that this gene is not functional. Many of the unique genes are found in the replication termination region (RTR or plasticity zone), an area of frequent symmetrical inversion events around the replication terminus shown to be a hotspot for genome variation in previous genome sequencing studies. In C.caviae, the RTR includes several loci of particular interest including a large toxin gene and evidence of ancestral insertion(s) of a bacteriophage. This toxin gene, not present in Chlamydia pneumoniae, is a member of the YopT effector family of type III-secreted cysteine proteases. One gene cluster (guaBA-add) in the RTR is much more similar to orthologs in Chlamydia muridarum than those in the phylogenetically closest species C.pneumoniae, suggesting the possibility of horizontal transfer of genes between the rodent-associated Chlamydiae. With most genes observed in the other chlamydial genomes represented, C.caviae provides a good model for the Chlamydiaceae and a point of comparison against the human atherosclerosis-associated C.pneumoniae. This crucial addition to the set of completed Chlamydiaceae genome sequences is enabling dissection of the roles played by niche-specific genes in these important bacterial pathogens.

Role of mobile DNA in the evolution of vancomycin-resistant Enterococcus faecalis.

The complete genome sequence of Enterococcus faecalis V583, a vancomycin-resistant clinical isolate, revealed that more than a quarter of the genome consists of probable mobile or foreign DNA. One of the predicted mobile elements is a previously unknown vanB vancomycin-resistance conjugative transposon. Three plasmids were identified, including two pheromone-sensing conjugative plasmids, one encoding a previously undescribed pheromone inhibitor. The apparent propensity for the incorporation of mobile elements probably contributed to the rapid acquisition and dissemination of drug resistance in the enterococci.

Whole-genome comparison of Mycobacterium tuberculosis clinical and laboratory strains.

Virulence and immunity are poorly understood in Mycobacterium tuberculosis. We sequenced the complete genome of the M. tuberculosis clinical strain CDC1551 and performed a whole-genome comparison with the laboratory strain H37Rv in order to identify polymorphic sequences with potential relevance to disease pathogenesis, immunity, and evolution. We found large-sequence and single-nucleotide polymorphisms in numerous genes. Polymorphic loci included a phospholipase C, a membrane lipoprotein, members of an adenylate cyclase gene family, and members of the PE/PPE gene family, some of which have been implicated in virulence or the host immune response. Several gene families, including the PE/PPE gene family, also had significantly higher synonymous and nonsynonymous substitution frequencies compared to the genome as a whole. We tested a large sample of M. tuberculosis clinical isolates for a subset of the large-sequence and single-nucleotide polymorphisms and found widespread genetic variability at many of these loci. We performed phylogenetic and epidemiological analysis to investigate the evolutionary relationships among isolates and the origins of specific polymorphic loci. A number of these polymorphisms appear to have occurred multiple times as independent events, suggesting that these changes may be under selective pressure. Together, these results demonstrate that polymorphisms among M. tuberculosis strains are more extensive than initially anticipated, and genetic variation may have an important role in disease pathogenesis and immunity.

Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440.

Pseudomonas putida is a metabolically versatile saprophytic soil bacterium that has been certified as a biosafety host for the cloning of foreign genes. The bacterium also has considerable potential for biotechnological applications. Sequence analysis of the 6.18 Mb genome of strain KT2440 reveals diverse transport and metabolic systems. Although there is a high level of genome conservation with the pathogenic Pseudomonad Pseudomonas aeruginosa (85% of the predicted coding regions are shared), key virulence factors including exotoxin A and type III secretion systems are absent. Analysis of the genome gives insight into the non-pathogenic nature of P. putida and points to potential new applications in agriculture, biocatalysis, bioremediation and bioplastic production.

Complete genome sequence of a virulent isolate of Streptococcus pneumoniae.

The 2,160,837-base pair genome sequence of an isolate of Streptococcus pneumoniae, a Gram-positive pathogen that causes pneumonia, bacteremia, meningitis, and otitis media, contains 2236 predicted coding regions; of these, 1440 (64%) were assigned a biological role. Approximately 5% of the genome is composed of insertion sequences that may contribute to genome rearrangements through uptake of foreign DNA. Extracellular enzyme systems for the metabolism of polysaccharides and hexosamines provide a substantial source of carbon and nitrogen for S. pneumoniae and also damage host tissues and facilitate colonization. A motif identified within the signal peptide of proteins is potentially involved in targeting these proteins to the cell surface of low-guanine/cytosine (GC) Gram-positive species. Several surface-exposed proteins that may serve as potential vaccine candidates were identified. Comparative genome hybridization with DNA arrays revealed strain differences in S. pneumoniae that could contribute to differences in virulence and antigenicity.

Complete genome sequence of Caulobacter crescentus.

The complete genome sequence of Caulobacter crescentus was determined to be 4,016,942 base pairs in a single circular chromosome encoding 3,767 genes. This organism, which grows in a dilute aquatic environment, coordinates the cell division cycle and multiple cell differentiation events. With the annotated genome sequence, a full description of the genetic network that controls bacterial differentiation, cell growth, and cell cycle progression is within reach. Two-component signal transduction proteins are known to play a significant role in cell cycle progression. Genome analysis revealed that the C. crescentus genome encodes a significantly higher number of these signaling proteins (105) than any bacterial genome sequenced thus far. Another regulatory mechanism involved in cell cycle progression is DNA methylation. The occurrence of the recognition sequence for an essential DNA methylating enzyme that is required for cell cycle regulation is severely limited and shows a bias to intergenic regions. The genome contains multiple clusters of genes encoding proteins essential for survival in a nutrient poor habitat. Included are those involved in chemotaxis, outer membrane channel function, degradation of aromatic ring compounds, and the breakdown of plant-derived carbon sources, in addition to many extracytoplasmic function sigma factors, providing the organism with the ability to respond to a wide range of environmental fluctuations. C. crescentus is, to our knowledge, the first free-living alpha-class proteobacterium to be sequenced and will serve as a foundation for exploring the biology of this group of bacteria, which includes the obligate endosymbiont and human pathogen Rickettsia prowazekii, the plant pathogen Agrobacterium tumefaciens, and the bovine and human pathogen Brucella abortus.

Complete genome sequence of Neisseria meningitidis serogroup B strain MC58.

The 2,272,351-base pair genome of Neisseria meningitidis strain MC58 (serogroup B), a causative agent of meningitis and septicemia, contains 2158 predicted coding regions, 1158 (53.7%) of which were assigned a biological role. Three major islands of horizontal DNA transfer were identified; two of these contain genes encoding proteins involved in pathogenicity, and the third island contains coding sequences only for hypothetical proteins. Insights into the commensal and virulence behavior of N. meningitidis can be gleaned from the genome, in which sequences for structural proteins of the pilus are clustered and several coding regions unique to serogroup B capsular polysaccharide synthesis can be identified. Finally, N. meningitidis contains more genes that undergo phase variation than any pathogen studied to date, a mechanism that controls their expression and contributes to the evasion of the host immune system.

Genome sequences of Chlamydia trachomatis MoPn and Chlamydia pneumoniae AR39.

The genome sequences of Chlamydia trachomatis mouse pneumonitis (MoPn) strain Nigg (1 069 412 nt) and Chlamydia pneumoniae strain AR39 (1 229 853 nt) were determined using a random shotgun strategy. The MoPn genome exhibited a general conservation of gene order and content with the previously sequenced C.trachomatis serovar D. Differences between C.trachomatis strains were focused on an approximately 50 kb 'plasticity zone' near the termination origins. In this region MoPn contained three copies of a novel gene encoding a >3000 amino acid toxin homologous to a predicted toxin from Escherichia coli O157:H7 but had apparently lost the tryptophan biosyntheis genes found in serovar D in this region. The C. pneumoniae AR39 chromosome was >99.9% identical to the previously sequenced C.pneumoniae CWL029 genome, however, comparative analysis identified an invertible DNA segment upstream of the uridine kinase gene which was in different orientations in the two genomes. AR39 also contained a novel 4524 nt circular single-stranded (ss)DNA bacteriophage, the first time a virus has been reported infecting C. pneumoniae. Although the chlamydial genomes were highly conserved, there were intriguing differences in key nucleotide salvage pathways: C.pneumoniae has a uridine kinase gene for dUTP production, MoPn has a uracil phosphororibosyl transferase, while C.trachomatis serovar D contains neither gene. Chromosomal comparison revealed that there had been multiple large inversion events since the species divergence of C.trachomatis and C.pneumoniae, apparently oriented around the axis of the origin of replication and the termination region. The striking synteny of the Chlamydia genomes and prevalence of tandemly duplicated genes are evidence of minimal chromosome rearrangement and foreign gene uptake, presumably owing to the ecological isolation of the obligate intracellular parasites. In the absence of genetic analysis, comparative genomics will continue to provide insight into the virulence mechanisms of these important human pathogens.

Optimized multiplex PCR: efficiently closing a whole-genome shotgun sequencing project.

A new method has been developed for rapidly closing a large number of gaps in a whole-genome shotgun sequencing project. The method employs multiplex PCR and a novel pooling strategy to minimize the number of laboratory procedures required to sequence the unknown DNA that falls in between contiguous sequences. Multiplex sequencing, a novel procedure in which multiple PCR primers are used in a single sequencing reaction, is used to interpret the multiplex PCR results. Two protocols are presented, one that minimizes pipetting and another that minimizes the number of reactions. The pipette optimized multiplex PCR method has been employed in the final phases of closing the Streptococcus pneumoniae genome sequence, with excellent results.

The self-fulfilling prophecy in close relationships: rejection sensitivity and rejection by romantic partners.

The authors hypothesized a self-fulfilling prophecy wherein rejection expectancies lead people to behave in ways that elicit rejection from their dating partners. The hypothesis was tested in 2 studies of conflict in couples: (a) a longitudinal field study where couples provided daily-diary reports and (b) a lab study involving behavioral observations. Results from the field study showed that high rejection-sensitive (HRS) people's relationships were more likely to break up than those of low rejection-sensitive (LRS) people. Conflict processes that contribute to relationship erosion were revealed for HRS women but not for HRS men. Following naturally occurring relationship conflicts, HRS women's partners were more rejecting than were LRS women's partners. The lab study showed that HRS women's negative behavior during conflictual discussions helped explain their partners' more rejecting postconflict responses.

Saphenous vein graft growth 13 years after coronary bypass in a child with Kawasaki disease.

The presumed limited growth potential of saphenous vein grafts has led many authorities to discourage their use in young children. We documented excellent growth and patency of a saphenous vein graft 13 years after operation in a 7-year-old child with coronary artery obstruction caused by Kawasaki disease.

Unusual case of prominent Chiari network trapped in the left atrium.

We describe a case of an unusually prominent Chiari network in a premature neonate who was evaluated with echocardiography. The network, which is an embryologic remnant, was extensive, mobile, and moved in and out of the right ventricle. Later, tissue strands passed the foramen ovale and ultimately became trapped in the left atrium. The differential diagnosis included thrombus, tumor, vegetation, and ruptured chordae.

Application of PCR for detection and identification of mycoplasma contamination in virus stocks.

A nested polymerase chain reaction (PCR) was used to detect and identify mycoplasma contaminants in viral stocks. The results of the PCR assay proved to be a sensitive and accurate indicator of the true status of the stock tested. Those samples positive by agar culture or Hoechst stain were also positive by PCR. Those samples that were inconclusive by Hoechst stain (10.05%) could be clearly determined to be mycoplasma positive or negative by PCR. The PCR assay also detected those fastidious species of mycoplasma that gave false negative results by the direct culture method. In many respects the PCR-based mycoplasma detection method described is superior to the agar culture and Hoechst staining detection methods. In this study, the PCR assay detected substantially more mycoplasma-positive viral stocks than did the agar culture assay. Due to its speed, sensitivity, and reliability, the PCR assay is of particular value in monitoring the process of removing mycoplasma from contaminated stocks. Furthermore, the PCR amplification products can be analyzed by restriction analysis to rapidly identify the species of the mycoplasma contaminating the stock tested.

Pharmacokinetics of the combination of fluvastatin and gemfibrozil.

High-risk patients with dyslipidemias resistant to diet and single-agent pharmacotherapy may require combination therapy to achieve target levels of low density lipoprotein, triglycerides, and high density lipoprotein. Combinations of fibrates and 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors are effective, but because of safety concerns related to myopathy and rhabdomyolysis, it is important to consider the possibility of pharmacokinetic interactions when such combinations are used. In this study, the area under the curve, maximum plasma concentration, and time to maximum concentration for fluvastatin and gemfibrozil are compared, when used alone and in combination, in patients with hyperlipidemia and either coronary or carotid atherosclerosis, or a family history of coronary artery disease. A total of 17 patients were studied in a random sequence, open-label, crossover study of fluvastatin at 20 mg twice daily, gemfibrozil at 600 mg twice daily, and the combination of the 2 drugs. No significant difference was observed in area under the curve, maximum plasma concentration, and time to maximum concentration when comparing the combination with each drug alone. These pharmacokinetic data add support to the clinical observations that the combination of fluvastatin and gemfibrozil is both effective and safe.

Molecular basis of the inhibition of gentamicin nephrotoxicity by daptomycin; an infrared spectroscopic investigation.

The lipopeptide daptomycin has been reported to reduce in vivo the nephrotoxicity of aminoglycoside antibiotics (Wood et al. (1989) Antimicrob. Agents Chemother. 33, 1280-1285; Beauchamp et al. (1990) Antimicrob. Agents Chemother. 34, 139-147). A recent dialysis study confirmed the existence of an electrostatic interaction between daptomycin and tobramycin (Couture et al. (1994) Antimicrob. Agents Chemother. 38, 742-749). The interaction of gentamicin with daptomycin and phosphatidylinositol (PI) dispersions was investigated by FTIR spectroscopy. We found no evidence of a direct interaction involving the neutralization of the aspartate groups of daptomycin by gentamicin and the amide I band of daptomycin did not reveal significant conformational changes of its peptidic moiety. On the other hand, daptomycin readily inserts within bilayers of PI, dimyristoylphosphatidylglycerol or dipalmitoylphosphatidylcholine, as judged from its influence on the fluidity of these bilayers. The incorporation of daptomycin into PI bilayers has no significant effect on the lipopeptide amide I band. Gentamicin also binds to PI bilayers and the associated modifications of the lipid bands are consistent with a tightening of the lipid network resulting from head group neutralization by gentamicin. The affinity of the aminoglycoside for PI is slightly increased in the presence of daptomycin, in agreement with the results of the dialysis study mentioned above. The lipid features indicate that its head group is still affected by gentamicin charges, but the thermotropic behavior of the hydrophobic portion becomes similar to that of the pure lipid. It is proposed that the contribution of daptomycin to the membrane charge density and its effect on the lipid packing both combine to counteract the inhibition of phospholipase activity due to aminoglycosides. Further work will attempt to determine how the peptide rings and gentamicin molecules are organized at the bilayer surface, how specific these interactions are and to confirm the influence of daptomycin on the phospholipid catabolism.

Long lag times and high velocities in the motility of natural assemblages of marine bacteria.

The motility characteristics of natural assemblages of coastal marine bacteria were examined. Initially, less than 10% of the bacteria were motile. A single addition of tryptic soy broth caused an increase in the motile fraction of cells but only after 7 to 12 h. Motility peaked at 15 to 30 h, when more than 80% of cells were motile. These results support the proposal that energy limits motility in the marine environment. Cell speeds changed more than an order of magnitude on timescales of milliseconds and hours. The maximum community speed was 144 (mu)m s(sup-1), and the maximum individual burst velocity was 407 (mu)m s(sup-1). In uniform medium, speed was an inverse function of tryptic soy broth concentration, declining linearly over 0.001 to 1.0%. In media where concentration gradients existed, the mean speed was a function of position in a spatial gradient, changing from 69 to 144 (mu)m s(sup-1) over as little as 15 to 30 (mu)m. The results suggest that marine bacteria are capable of previously undescribed quick shifts in speed that may permit the bacteria to rapidly detect and keep up with positional changes in small nutrient sources. These high speeds and quick shifts may reflect the requirements for useful motility in a turbulent ocean.