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Tertiary Structure of Bacterial Murein: the Scaffold Model. Boris A. Dmitriev, 2003.Although the chemical structure and physical properties of peptidoglycan have been elucidated for some time, the precise three-dimensional organization of murein has remained elusive . Earlier published computer simulations of the bacterial murein architecture modeled peptidoglycan strands in either a regular (D . Pink, J . Moeller, B . Quinn, M . Jericho, and T . Beveridge, J . Bacteriol . 182: 5925-5930, 2000) or an irregular (A . Koch, J . Theor . Biol . 204: 533-541, 2000) parallel orientation with respect to the plasma membrane . However, after integrating published experimental data on glycan chain length distribution and the degree of peptide side chain cross-linking into this computer simulation, we now report that the proposed planar network of murein appears largely dysfunctional . In contrast, a scaffold model of murein architecture, which assumes that glycan strands extend perpendicularly to the plasma membrane, was found to accommodate published experimental evidence and yield a viable stress-bearing matrix . Moreover, this model is in accordance with the well-established principle of murein assembly in vivo, i.e., sequential attachment of strands to the preexisting structure . For the first time, the phenomenon of division plane alternation in dividing bacteria can be reconciled with a computer model of the molecular architecture of murein . Incidence of Antibiotic Resistance in Campylobacter jejuni Isolated in Alberta, Canada, from 1999 to 2002, with Special Reference to tet(O)-Mediated Tetracycline Resistance. Amera Gibreel, 2004.Of 203 human clinical isolates of Campylobacter jejuni from Alberta, Canada (1999 to 2002), 101 isolates (50%) were resistant to at least 64 µg of tetracycline/ml, with four isolates exhibiting higher levels of tetracycline resistance (512 µg/ml) . In total, the MICs for 37% of tetracycline-resistant isolates (256 to 512 µg/ml) were higher than those previously reported in C . jejuni (64 to 128 µg/ml) . In the tetracycline-resistant clinical isolates, 67% contained plasmids and all contained the tet(O) gene . Four isolates resistant to high levels of tetracycline (MIC = 512 µg/ml) contained plasmids carrying the tet(O) gene, which could be transferred to other isolates of C . jejuni . The tetracycline MICs for transconjugants were comparable to those of the donors . Cloning of tet(O) from the four high-level tetracycline-resistant isolates conferred an MIC of 32 µg/ml for Escherichia coli DH5  . In contrast, transfer to a strain of C . jejuni by using mobilization conferred an MIC of 128 µg/ml . DNA sequence analysis determined that the tet(O) genes encoding lower MICs (64 to 128 µg/ml) were identical to one other, although the tet(O) genes encoding a 512-µg/ml MIC demonstrated several nucleotide substitutions . The quinolone resistance determining region of four ciprofloxacin-resistant isolates (2%) was analyzed, and resistance was associated with a chromosomal mutation in the gyrA gene resulting in a Thr-86-Ile substitution . In addition, six kanamycin-resistant isolates contained large plasmids that carry the aphA-3 marker coding for 3'-aminoglycoside phosphotransferase . Resistance to erythromycin was not detected in 203 isolates . In general, resistance to most antibiotics in C . jejuni remains low, except for resistance to tetracycline, which has increased from about 8 to 50% over the past 20 years .
The Glycolytic Flux in Escherichia coli Is Controlled by the Demand for ATP. Brian J. Koebmann, 2002.The nature of the control of glycolytic flux is one of the central, as-yet-uncharacterized issues in cellular metabolism . We developed a molecular genetic tool that specifically induces ATP hydrolysis in living cells without interfering with other aspects of metabolism . Genes encoding the F1 part of the membrane-bound (F1F0) H+-ATP synthase were expressed in steadily growing Escherichia coli cells, which lowered the intracellular [ATP]/[ADP] ratio . This resulted in a strong stimulation of the specific glycolytic flux concomitant with a smaller decrease in the growth rate of the cells . By optimizing additional ATP hydrolysis, we increased the flux through glycolysis to 1.7 times that of the wild-type flux . The results demonstrate why attempts in the past to increase the glycolytic flux through overexpression of glycolytic enzymes have been unsuccessful: the majority of flux control (>75%) resides not inside but outside the pathway, i.e., with the enzymes that hydrolyze ATP . These data further allowed us to answer the question of whether catabolic or anabolic reactions control the growth of E . coli . We show that the majority of the control of growth rate resides in the anabolic reactions, i.e., the cells are mostly "carbon" limited . Ways to increase the efficiency and productivity of industrial fermentation processes are discussed . The Viable but Nonculturable State and Starvation Are Different Stress Responses of Enterococcus faecalis, as Determined by Proteome Analysis. Sabina Heim, 2002.The protein expression patterns of exponentially growing, starved, and viable but nonculturable (VBNC) Enterococcus faecalis cells were analyzed to establish whether differences exist between the VBNC state and other stress responses . The results indicate that the protein profile of VBNC cells differs from that of either starved or exponentially growing bacteria . This demonstrates that the VBNC state is a distinct physiological phase within the life cycle of E . faecalis, which is activated in response to multiple environmental stresses . Vector Systems Allowing Efficient Autonomous or Integrative Gene Cloning in Micromonospora sp . Strain 40027. Xiaohua Li, 2003.Vector systems allowing autonomous or site-specific integrative gene cloning were developed for Micromonospora sp . strain 40027, a producer of the antibiotic fortimicin A . The autonomous system depends on the discovery of a low-copy-number, self-transmissible covalently closed circular plasmid, pJTU112 (ca . 14.1 kb), which was shown to be present in the progenitor strain in both integrated and autonomous states . The copy numbers of both wild-type pJTU112 and three derivatives of it can be amplified at least sixfold by addition of streptomycin to the culture medium . The integrative system was developed by the use of a pBR322-derived Escherichia coli plasmid vector, pSET152, mediated by the attP site of the Streptomyces phage  C31 . Both vectors can be transferred by conjugation from E . coli into Micromonospora sp . strain 40027 . The heterologous cloning and expression of the dnd gene cluster originating from Streptomyces lividans 1326 into Micromonospora sp . strain 40027 demonstrated the use of the two systems .
Community Composition and Functioning of Denitrifying Bacteria from Adjacent Meadow and Forest Soils. J. J. Rich, 2003.We investigated communities of denitrifying bacteria from adjacent meadow and forest soils . Our objectives were to explore spatial gradients in denitrifier communities from meadow to forest, examine whether community composition was related to ecological properties (such as vegetation type and process rates), and determine phylogenetic relationships among denitrifiers . nosZ, a key gene in the denitrification pathway for nitrous oxide reductase, served as a marker for denitrifying bacteria . Denitrifying enzyme activity (DEA) was measured as a proxy for function . Other variables, such as nitrification potential and soil C/N ratio, were also measured . Soil samples were taken along transects that spanned meadow-forest boundaries at two sites in the H . J . Andrews Experimental Forest in the Western Cascade Mountains of Oregon . Results indicated strong functional and structural community differences between the meadow and forest soils . Levels of DEA were an order of magnitude higher in the meadow soils . Denitrifying community composition was related to process rates and vegetation type as determined on the basis of multivariate analyses of nosZ terminal restriction fragment length polymorphism profiles . Denitrifier communities formed distinct groups according to vegetation type and site . Screening 225 nosZ clones yielded 47 unique denitrifying genotypes; the most dominant genotype occurred 31 times, and half the genotypes occurred once . Several dominant and less-dominant denitrifying genotypes were more characteristic of either meadow or forest soils . The majority of nosZ fragments sequenced from meadow or forest soils were most similar to nosZ from the Rhizobiaceae group in  -Proteobacteria species . Denitrifying community composition, as well as environmental factors, may contribute to the variability of denitrification rates in these systems .
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