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Energy-Generating Enzymes of Burkholderia cepacia and Their Interactions with Macrophages. Vasu Punj, 2003.We previously demonstrated that several clinical and environmental isolates of Burkholderia cepacia secreted ATP-utilizing enzymes to the medium; the secretion of these enzymes by cystic fibrosis lung isolate strain 38 was shown to be greatly enhanced in the presence of  2-macroglobulin .
Fractionation of the growth medium of cystic fibrosis isolate strain
71 belonging to genomovar I demonstrated the presence of two
additional proteins, homologues of Pseudomonas aeruginosa
azurin and cytochrome c551, which are normally
involved in electron transfer during denitrification . A Q-Sepharose
column flowthrough fraction of the growth medium of B . cepacia
strain 71 enriched with the azurin and cytochrome c551
homologues triggered apoptosis in macrophages and mast cells, leading
to their death . Incubation of the Q-Sepharose column flowthrough
fraction with antiazurin and anti-cytochrome c551
antibodies greatly reduced cell death . We cloned and hyperexpressed a
gene from B . cepacia strain 71 that encodes the homologue of
P . aeruginosa azurin . Such azurin homologues were detected in
the growth medium of several strains belonging to genomovars I, III,
and VI but not in the growth medium of strains belonging to other
genomovars . The growth medium of the strains that elaborated the
azurin homologue had high cytotoxicity towards macrophages . Purified
azurin homologue was shown to induce apoptosis in macrophages in a
caspase-dependent manner and was localized in both the cytosol and
nucleus when incubated with or microinjected into macrophages . This
is an interesting example of the interaction of a bacterial protein
normally involved in cellular energetics with macrophages to effect
their cell death .
Genome Sequence of Yersinia pestis KIM. Wen Deng, 2002.We present the complete genome sequence of Yersinia pestis KIM, the etiologic agent of bubonic and pneumonic plague . The strain KIM, biovar Mediaevalis, is associated with the second pandemic, including the Black Death . The 4.6-Mb genome encodes 4,198 open reading frames (ORFs) . The origin, terminus, and most genes encoding DNA replication proteins are similar to those of Escherichia coli K-12 . The KIM genome sequence was compared with that of Y . pestis CO92, biovar Orientalis, revealing homologous sequences but a remarkable amount of genome rearrangement for strains so closely related . The differences appear to result from multiple inversions of genome segments at insertion sequences, in a manner consistent with present knowledge of replication and recombination . There are few differences attributable to horizontal transfer . The KIM and E . coli K-12 genome proteins were also compared, exposing surprising amounts of locally colinear "backbone," or synteny, that is not discernible at the nucleotide level . Nearly 54% of KIM ORFs are significantly similar to K-12 proteins, with conserved housekeeping functions . However, a number of E . coli pathways and transport systems and at least one global regulator were not found, reflecting differences in lifestyle between them . In KIM-specific islands, new genes encode candidate pathogenicity proteins, including iron transport systems, putative adhesins, toxins, and fimbriae . Two Opines Control Conjugal Transfer of an Agrobacterium Plasmid by Regulating Expression of Separate Copies of the Quorum-Sensing Activator Gene traR. Philippe Oger, 2002.Conjugal transfer of Ti plasmids from Agrobacterium spp . is controlled by a hierarchical regulatory system designed to sense two environmental cues . One signal, a subset of the opines produced by crown gall tumors initiated on plants by the pathogen, serves to induce production of the second, an acyl-homoserine lactone quorum-sensing signal, the quormone, produced by the bacterium itself . This second signal activates TraR, and this transcriptional activator induces expression of the tra regulon . Opines control transfer because the traR gene is a member of an operon the expression of which is regulated by the conjugal opine . Among the Ti plasmid systems studied to date, only one of the two or more opine families produced by the associated tumor induces transfer . However, two chemically dissimilar opines, nopaline and agrocinopines A and B, induce transfer of the opine catabolic plasmid pAtK84b found in the nonpathogenic Agrobacterium radiobacter isolate K84 . In this study we showed that this plasmid contains two copies of traR, and each is associated with a different opine-regulated operon . One copy, traRnoc, is the last gene of the nox operon and was induced by nopaline but not by agrocinopines A and B . Mutating traRnoc abolished induction of transfer by nopaline but not by the agrocinopines . A mutation in ocd, an upstream gene of the nox operon, abolished utilization of nopaline and also induction of transfer by this opine . The second copy, traRacc, is located in an operon of four genes and was induced by agrocinopines A and B but not by nopaline . Genetic analysis indicated that this gene is required for induction of transfer by agrocinopines A and B but not by nopaline . pAtK84b with mutations in both traR genes was not induced for transfer by either opine . However, expression of a traR gene in trans to this plasmid resulted in opine-independent transfer . The association of traRnoc with nox is unique, but the operon containing traRacc is related to the arc operons of pTiC58 and pTiChry5, two Ti plasmids inducible for transfer by agrocinopines A-B and C-D, respectively . We conclude that pAtK84b codes for two independently functioning copies of traR, each regulated by a different opine, thus accounting for the activation of the transfer system of this plasmid by the two opine types . Oligonucleotide Microarray for the Study of Functional Gene Diversity in the Nitrogen Cycle in the Environment. Gaspar Taroncher-Oldenburg, 2003.The analysis of functional diversity and its dynamics in the environment is essential for understanding the microbial ecology and biogeochemistry of aquatic systems . Here we describe the development and optimization of a DNA microarray method for the detection and quantification of functional genes in the environment and report on their preliminary application to the study of the denitrification gene nirS in the Choptank River-Chesapeake Bay system . Intergenic and intragenic resolution constraints were determined by an oligonucleotide (70-mer) microarray approach . Complete signal separation was achieved when comparing unrelated genes within the nitrogen cycle (amoA, nifH, nirK, and nirS) and detecting different variants of the same gene, nirK, corresponding to organisms with two different physiological modes, ammonia oxidizers and denitrifying halobenzoate degraders . The limits of intragenic resolution were investigated with a microarray containing 64 nirS sequences comprising 14 cultured organisms and 50 clones obtained from the Choptank River in Maryland . The nirS oligonucleotides covered a range of sequence identities from approximately 40 to 100% . The threshold values for specificity were determined to be 87% sequence identity and a target-to-probe perfect match-to-mismatch binding free-energy ratio of 0.56 . The lower detection limit was 10 pg of DNA (equivalent to approximately 107 copies) per target per microarray . Hybridization patterns on the microarray differed between sediment samples from two stations in the Choptank River, implying important differences in the composition of the denitirifer community along an environmental gradient of salinity, inorganic nitrogen, and dissolved organic carbon . This work establishes a useful set of design constraints (independent of the target gene) for the implementation of functional gene microarrays for environmental applications .
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