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Archives of Microbiology (v.183, #3)
Metabolic monitoring by bacterial mRNAs by Wade C. Winkler (pp. 151-159).
There is growing appreciation for diversity in the strategies that bacteria utilize in regulating gene expression. Bacteria must be able to respond in different ways to different stresses and thus require unique regulatory solutions for the physiological challenges they encounter. Recent data indicate that bacteria commonly employ a variety of posttranscriptional regulatory mechanisms to coordinate expression of their genes. In many instances, RNA structures embedded at the 5′ ends of mRNAs are utilized to sense particular metabolic cues and regulate the encoded genes. These RNA elements are likely to range in structural sophistication, from short sequences recognized by RNA-binding proteins to complex shapes that fold into high-affinity receptors for small organic molecules. Enough examples of RNA-mediated genetic strategies have been found that it is becoming useful to view this overall mode of regulatory control at a genomic level. Eventually, a complete picture of bacterial gene regulation within a single bacterium, from control at transcription initiation to control of mRNA stability, will emerge. But for now, this article seeks to provide a brief overview of the known categories of RNA-mediated genetic mechanisms within the bacterium Bacillus subtilis, with the expectation that it is representative of bacteria as a whole.
Keywords: RNA; Aptamer; Riboswitch; Transcription attenuation; Noncoding regulation; BacteriaBacillus subtilis
The nuclei of Giardia lamblia—new ultrastructural observations by Marlene Benchimol (pp. 160-168).
Giardia lamblia is a parasite possessing a complex cytoskeleton and an unusual morphology of bearing two nuclei. Here, the interphasic nuclei of trophozoites, using field emission scanning electron microscopy, routine scanning and transmission electron microscopy, immunocytochemistry, and 3D reconstruction, are presented. An approach using plasma-membrane extraction allowed the observation of the two nuclei still attached in their original positions. The observations are as follows: (1) Giardia nuclei and cytoskeleton were studied in demembranated cells by routine scanning electron microscopy and field emission; (2) both nuclei are anchored to basal bodies of the anterior flagella and to the descending posterior-lateral and ventral flagella, at the right and left nuclei, respectively, in cells attached by its ventral disc; (3) this attachment occurs by proteinaceous links, which were labeled by anti-actin and anti-centrin but not by anti-dynein or anti-tubulin antibodies; (4) fibrilar connections between the nuclei and the disc were also observed; and (5) nuclei exhibited a pendular movement when living cells were treated with cytochalasin, although the nuclei were still connected by their anterior region. Our analysis indicated that the nuclei have a defined position, and fibrils perform an anchoring system. This raises the possibility of a mechanism for nuclei-fidelity migration during mitosis.
Keywords: Giardia lambliaNuclei; Basal body; SEM; FESEM; Axonemes
A new amplification target for PCR–RFLP detection and identification of Chlamydiaceae species by Vladimir V. Demkin; Andrey L. Zimin (pp. 169-175).
The family Chlamydiaceae contains nine species pathogenic to humans and animals, but their routine identification is hampered by inadequate detection methods. In an attempt to find a new region for PCR detection and discrimination of the Chlamydiaceae species, the 3′ end of the omp2 gene of Chlamydiaceae has been examined. Since sequence data for this part of the genes of Chlamydophila felis and Chlamydia suis had not been available, the near full length of the omp2 genes of these species were cloned and sequenced. Consensus primers enabling amplification of a previously untargeted region spanning 1,030 bp at the 3′ end of the gene were designed. Discrimination of all nine Chlamydiaceae species was achieved via RFLP analysis of the amplicons with RsaI and HinfI or RsaI and TaqI endonucleases or via electrophoretic mobility analysis of the RsaI restriction fragments in agarose gel with bisbenzimide-PEG. Intraspecies uniformity of the RFLP patterns was evaluated by the typing of reference strains, isolates of human and animal origin from culture collections, and clinical specimens, and by computer analysis of GenBank sequences. The 3′ end of the omp2 gene was shown to be an appropriate marker region suitable for rapid identification of Chlamydiaceae species and can be used for characterization of collection strains and new isolates in taxonomic, epidemiological, and clinical purposes.
Keywords: Chlamydiaceae; Outer membrane proteins; Species specificity; Classification; DNA primers; Polymerase chain reaction
Identification and functional characterisation of genes and corresponding enzymes involved in carnitine metabolism of Proteus sp. by Claudia Engemann; Thomas Elssner; Sven Pfeifer; Carsten Krumbholz; Thomas Maier; Hans-Peter Kleber (pp. 176-189).
Enzymes involved in carnitine metabolism of Proteus sp. are encoded by the cai genes organised as the caiTABCDEF operon. The complete operon could be sequenced from the genomic DNA of Proteus sp. Amino acid sequence similarities and/or enzymatic analysis confirmed the function assigned to each protein involved in carnitine metabolism. CaiT was suggested to be an integral membrane protein responsible for the transport of betaines. The caiA gene product was shown to be a crotonobetainyl-CoA reductase catalysing the irreversible reduction of crotonobetainyl-CoA to γ-butyrobetainyl-CoA. CaiB and CaiD were identified to be the two components of the crotonobetaine hydrating system, already described. CaiB and caiD were cloned and expressed in Escherichia coli. After purification of both proteins, their individual enzymatic functions were solved. CaiB acts as betainyl-CoA transferase specific for carnitine, crotonobetaine, γ-butyrobetaine and its CoA derivatives. Transferase reaction proceeds, following a sequential bisubstrate mechanism. CaiD was identified to be a crotonobetainyl-CoA hydratase belonging to the crotononase superfamily. Because of amino acid sequence similarities, CaiC was suggested to be a betainyl-CoA ligase. Taken together, these results show that the metabolism of carnitine and crotonobetaine in Proteus sp. proceeds at the CoA level.
Keywords: Carnitine; CoA transferase; Enoyl-CoA hydrataseProteus sp.; Reductase
Large reallocations of carbon, nitrogen, and photosynthetic reductant among phycobilisomes, photosystems, and Rubisco during light acclimation in Synechococcus elongatus strain PCC7942 are constrained in cells under low environmental inorganic carbon by Tyler D.B. MacKenzie; Jeanette M. Johnson; Amanda M. Cockshutt; Robert A. Burns; Douglas A. Campbell (pp. 190-202).
Synechococcus elongatus strain PCC7942 cells were grown in high or low environmental concentrations of inorganic C (high-Ci, low-Ci) and subjected to a light shift from 50 µmol m−2 s−1 to 500 µmol m−2 s−1. We quantified photosynthetic reductant (O2 evolution) and molar cellular contents of phycobilisomes, PSII, PSI, and ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) through the light shift. Upon the increase in light, small initial relative decreases in phycobilisomes per cell resulted from near cessation of phycobilisome synthesis and their dilution into daughter cells. Thus, allocation of reductant to phycobilisome synthesis dropped fivefold from pre- to post-light shift. The decrease in phycobilisome synthesis liberated enough material and reductant to allow a doubling of Rubisco and up to a sixfold increase in PSII complexes per cell. Low-Ci cells had smaller initial phycobilisome pools and upon increased light; their reallocation of reductant from phycobilisome synthesis may have limited the rate and extent of light acclimation, compared to high-Ci cells. Acclimation to increased light involved large reallocations of C, N, and reductant among different components of the photosynthetic apparatus, but total allocation to the apparatus was fairly stable at ca. 50% of cellular N, and drew 25–50% of reductant from photosynthesis.
Keywords: Carbon-concentrating mechanism; Macromolecular acclimation; Photosynthetic acclimation; Resource allocation
Sensitivity of dark mutants of various strains of luminescent bacteria to reactive oxygen species by Robert Łyżeń; Grzegorz Węgrzyn (pp. 203-208).
Recent studies indicated that bioluminescence of the marine bacterium Vibrio harveyi may both stimulate DNA repair and contribute to detoxification of deleterious oxygen derivatives. Therefore, it was also proposed that these reactions can be considered biological roles of bacterial luminescence and might act as evolutionary drives in development of luminous systems. However, experimental evidence for the physiological role of luciferase in protection of cells against oxidative stress has been demonstrated only in one bacterial species, raising the question whether this is a specific or a more general phenomenon. Here we demonstrate that in the presence of various oxidants (hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide and ferrous ions) growth of dark mutants of different strains of Vibrio fischeri and Photobacterium leiognathi is impaired relative to wild-type bacteria, though to various extents. Deleterious effects of oxidants on the mutants could be reduced (with different efficiency) by addition of antioxidants, A-TEMPO or 4OH-TEMPO. These results support the hypotheses that (1) activities of bacterial luciferases may detoxify deleterious oxygen derivatives, and (2) significantly different efficiencies of this reaction are characteristic for various luciferases.
Keywords: Bacterial luciferases; Oxidative stress; AntioxidantsVibrio fischeriPhotobacterium leiognathi
Effects of over-expression of the regulatory enzymes DraT and DraG on the ammonium-dependent post-translational regulation of nitrogenase reductase in Azospirillum brasilense by Luciano F. Huergo; Emanuel M. Souza; Maria B. R. Steffens; M. Geoffrey. Yates; Fábio O. Pedrosa; Leda S. Chubatsu (pp. 209-217).
Nitrogen fixation in Azospirillum brasilense is regulated at transcriptional and post-translational levels. Post-translational control occurs through the reversible ADP-ribosylation of dinitrogenase reductase (Fe Protein), mediated by the dinitrogenase reductase ADP-ribosyltransferase (DraT) and dinitrogenase reductase glycohydrolase (DraG). Although the DraT and DraG activities are regulated in vivo, the molecules responsible for such regulation remain unknown. We have constructed broad-host-range plasmids capable of over-expressing, upon IPTG induction, the regulatory enzymes DraT and DraG as six-histidine-N-terminal fused proteins (His). Both DraT-His and DraG-His are functional in vivo. We have analyzed the effects of DraT-His and DraG-His over-expression on the post-translational modification of Fe Protein. The DraT-His over-expression led to Fe Protein modification in the absence of ammonium addition, while cells over-expressing DraG-His showed only partial ADP-ribosylation of Fe Protein by adding ammonium. These results suggest that both DraT-His and DraG-His lose their regulation upon over-expression, possible by titrating out negative regulators.
Keywords: Azospirillum brasilenseDraT; DraG; Nitrogen fixation
Light-dependent expression of superoxide dismutase from cyanobacterium Synechocystis sp. strain PCC 6803 by Jae-Hyun Kim; Kyong Hoon Suh (pp. 218-223).
The oxygenic phototrophic cyanobacterium Synechocystis sp. strain PCC 6803 inevitably evolves superoxide during photosynthesis. Synechocystis 6803 contains only one type of superoxide dismutase, designated as SodB; therefore, this protein plays an important role in preventing oxidative damages caused by light. Because there was no direct evidence that SodB in Synechocystis 6803 could be regulated by light, the relationship between SodB and light was investigated in the present study. The activity of SodB from the cells grown in continuous light culture was about 3.5-fold higher than that from the cells cultivated in continuous dark. Illumination maximally activated SodB within 12 h. The level of sodB mRNA increased 12-fold by light, and that of SodB protein proportionally. Therefore, the expression and activity of SodB from Synechocystis 6803 were dependent on the light.
Keywords: Synechocystis sp. strain PCC 6803; Superoxide dismutase; SodB; Oxidative stress; Reactive oxygen species; Photoactivation
Unique regulation of glyoxalase I activity during osmotic stress response in the fission yeast Schizosaccharomyces pombe: neither the mRNA nor the protein level of glyoxalase I increase under conditions that enhance its activity by Yoshifumi Takatsume; Shingo Izawa; Yoshiharu Inoue (pp. 224-227).
Glyoxalase I is a ubiquitous enzyme that catalyzes the conversion of methylglyoxal, a toxic 2-oxoaldehyde derived from glycolysis, to S-D-lactoylglutathione. The activity of glyoxalase I in the fission yeast Schizosaccharomyces pombe was increased by osmotic stress induced by sorbitol. However, neither the mRNA levels of its structural gene nor its protein levels increased under the same conditions. Cycloheximide blocked the induction of glyoxalase I activity in cells exposed to osmotic stress. In addition, glyoxalase I activity was increased in stress-activated protein kinase-deficient mutants (wis1 and spc1). We present evidence for the post-translational regulation of glyoxalase I by osmotic stress in the fission yeast.
Keywords: Glyoxalase I; Methylglyoxal; Osmotic stress; MAP kinaseSchizosaccharomyces pombe