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Genomic and Genetic Analysis of Bordetella Bacteriophages Encoding Reverse Transcriptase-Mediated Tropism-Switching Cassettes. Minghsun Liu, 2004.Liu et al . recently described a group of related temperate bacteriophages that infect Bordetella subspecies and undergo a unique template-dependent,reverse transcriptase-mediated tropism switching phenomenon[Liu et al., Science 295: 2091-2094, 2002] . Tropism switchingresults from the introduction of single nucleotide substitutionsat defined locations in the VR1 [variable region 1] segmentof the mtd [major tropism determinant] gene, which determinesspecificity for receptors on host bacteria . In this report,we describe the complete nucleotide sequences of the 42.5- to 42.7-kb double-stranded DNA genomes of three related phage isolates and characterize two additional regions of variability . Forty-nine coding sequences were identified . Of these coding sequences, bbp36 contained VR2 [variable region 2], which is highly dynamic and consists of a variable number of identical 19-bp repeats separated by one of three 5-bp spacers, and bpm encodes a DNA adenine methylase with unusual site specificity and a homopolymer tract that functions as a hotspot for frameshift mutations. Morphological and sequence analysis suggests that these Bordetella phage are genetic hybrids of P22 and T7 family genomes, lending further support to the idea that regions encoding protein domains, single genes, or blocks of genes are readily exchanged between bacterial and phage genomes . Bordetella bacteriophages are capable of transducing genetic markers in vitro, and by using animal models, we demonstrated that lysogenic conversion can take placein the mouse respiratory tract during infection. Comparative Effects of Ciprofloxacin and Ceftazidime on Cytokine Production in Patients with Severe Sepsis Caused by Gram-Negative Bacteria. C. A. Gogos, 2004.In the present study the effect of ciprofloxacin versus ceftazidime on concentrations of pro- and anti-inflammatory cytokines in the sera of patients with severe sepsis was evaluated . The study included 58 previously healthy patients suffering from severe sepsis caused by gram-negative bacteria, treated with either ciprofloxacin or ceftazidime after thorough clinical and microbiological evaluation and followed up for clinical outcome . Levels of the proinflammatory cytokines tumor necrosis factor alpha (TNF-  ), interleukin-1b (IL-1b), IL-6, and IL-8 and of the anti-inflammatory cytokine IL-10, as well as of IL-1 receptor antagonist and soluble TNF receptors I and II, in serum were measured at baseline and 24 and 48 h after the first antimicrobial dose . Mean SAPS-II scores, development of septic shock, and mortality rates were similar in the two groups (43.2 ± 9.2, 21.4%, and 14.3% in the ceftazidime group versus 49.8 ± 11.3, 20%, and 13.3% in the ciprofloxacin group) . Serum TNF- and IL-6 levels at 24 and 48 h were significantly lower in the ciprofloxacin group, while the IL-10/TNF- ratio was significantly higher, than those for the ceftazidime group . Among patients with high baseline TNF- levels, there were significant increases in the IL-10/TNF- ratio at both 24 and 48 h over that at admission for the ciprofloxacin group, while no differences were noted in the ceftazidime group . These results indicate that ciprofloxacin may have an immunomodulatory effect on septic patients by attenuating the proinflammatory response, while there is no evidence that differences in the cytokines measured have any impact on the final outcome .
Isolation and Phylogenetic Analysis of Novel Viruses Infecting the Phytoplankton Phaeocystis globosa (Prymnesiophyceae). C. P. D. Brussaard, 2004.Viruses infecting the harmful bloom-causing alga Phaeocystis globosa (Prymnesiophyceae) were readily isolated from Dutch coastal waters (southern North Sea) in 2000 and 2001 . Our data show a large increase in the abundance of putative P . globosa viruses during blooms of P . globosa, suggesting that viruses are an important source of mortality for this alga . In order to examine genetic relatedness among viruses infecting P . globosa and other phytoplankton, DNA polymerase gene (pol) fragments were amplified and the inferred amino acid sequences were phylogenetically analyzed . The results demonstrated that viruses infecting P . globosa formed a closely related monophyletic group within the family Phycodnaviridae, with at least 96.9% similarity to each other . The sequences grouped most closely with others from viruses that infect the prymnesiophyte algae Chrysochromulina brevifilum and Chrysochromulina strobilus . Whether the P . globosa viruses belong to the genus Prymnesiovirus or form a separate group needs further study . Our data suggest that, like their phytoplankton hosts, the Chrysochromulina and Phaeocystis viruses share a common ancestor and that these prymnesioviruses and their algal host have coevolved . Metal Ion Dependence of Recombinant Escherichia coli Allantoinase. Scott B. Mulrooney, 2003.Allantoinase is a suspected dinuclear metalloenzyme that catalyzes the hydrolytic cleavage of the five-member ring of allantoin (5-ureidohydantoin) to form allantoic acid . Recombinant Escherichia coli allantoinase purified from overproducing cultures amended with 2.5 mM zinc, 1 mM cobalt, or 1 mM nickel ions was found to possess  1.4 Zn, 0.0 Co, 0.0 Ni, and 0.4 Fe; 0.1 Zn, 1.0 Co, 0.0 Ni, and 0.2 Fe; and 0.0 Zn, 0.0 Co, 0.6 Ni, and 0.1 Fe per subunit, respectively, whereas protein obtained from nonamended cultures contains near stoichiometric levels of iron . We conclude that allantoinase is incompletely activated in the recombinant cells, perhaps due to an insufficiency of a needed accessory protein . Enzyme isolated from nonsupplemented cultures possesses very low activity (kcat = 34.7 min-1) compared to the zinc-, cobalt-, and nickel-containing forms of allantoinase (kcat values of 5,000 and 28,200 min-1 and 200 min-1, respectively) . These rates and corresponding Km values (17.0, 19.5, and 80 mM, respectively) are significantly greater than those that have been reported previously . Absorbance spectroscopy of the cobalt species reveals a band centered at 570 nm consistent with five-coordinate geometry . Dithiothreitol is a competitive inhibitor of the enzyme, with significant Ki differences for the zinc and cobalt species (237 and 795 µM, respectively) . Circular dichroism spectroscopy revealed that the zinc enzyme utilizes only the S isomer of allantoin, whereas the cobalt allantoinase prefers the S isomer, but also hydrolyzes the R isomer at about 1/10 the rate . This is the first report for metal content of allantoinase from any source .
Genome-Scale Analysis of the Uses of the Escherichia coli Genome: Model-Driven Analysis of Heterogeneous Data Sets. Timothy E. Allen, 2003.The recent availability of heterogeneous high-throughput data types has increased the need for scalable in silico methods with which to integrate data related to the processes of regulation, protein synthesis, and metabolism . A sequence-based framework for modeling transcription and translation in prokaryotes has been established and has been extended to study the expression state of the entire Escherichia coli genome . The resulting in silico analysis of the expression state highlighted three facets of gene expression in E . coli: (i) the metabolic resources required for genome expression and protein synthesis were found to be relatively invariant under the conditions tested; (ii) effective promoter strengths were estimated at the genome scale by using global mRNA abundance and half-life data, revealing genes subject to regulation under the experimental conditions tested; and (iii) large-scale genome location-dependent expression patterns with approximately 600-kb periodicity were detected in the E . coli genome based on the 49 expression data sets analyzed . These results support the notion that a structured model-driven analysis of expression data yields additional information that can be subjected to commonly used statistical analyses . The integration of heterogeneous genome-scale data (i.e., sequence, expression data, and mRNA half-life data) is readily achieved in the context of an in silico model . Wide Geographic Distribution of Bacteriophages That Lyse the Same Indigenous Freshwater Isolate (Sphingomonas sp . Strain B18). Arite Wolf, 2003.An indigenous freshwater bacterium (Sphingomonas sp . strain B18) from Lake Plußsee (Schleswig-Holstein, Germany) was used to isolate 44 phages from 13 very different freshwater and brackish habitats in distant geographic areas . This bacterial strain was very sensitive to a broad spectrum of phages from different aquatic environments . Phages isolated from geographically distant aquatic habitats, but also those from the same sample, were diverse with respect to morphology and restriction pattern . Some phages were widely distributed, while different types coexisted in the same sample . It was concluded that phages could be a major factor in shaping the structure of bacterial communities and maintaining a high bacterial diversity .
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