|
|
|
|
|
|
mRNA Secondary Structure Modulates Translation of Tat-Dependent Formate Dehydrogenase N. Claire Punginelli, 2004.Formate dehydrogenase N [FDH-N] of Escherichia coli is a membrane-bound enzyme comprising FdnG, FdnH, and FdnI subunits organized inan [  ß ]3
configuration . The FdnG subunit carries aTat-dependent signal
peptide, which localizes the protein complexto the periplasmic side
of the membrane . We noted that substitutionof the first arginine [R5]
in the twin arginine signal sequenceof FdnG for a variety of other
amino acids resulted in a dramatic[up to 60-fold] increase in the
levels of protein synthesized.Bioinformatic analysis suggested that
the mRNA specifying thefirst 17 codons of fdnG forms a stable
stem-loop structure.A detailed mutational analysis has demonstrated
the importanceof this mRNA stem-loop in modulating FDH-N
translation.
Genotype and Phenotype Patterns of Human Immunodeficiency Virus Type 1 Resistance to Enfuvirtide during Long-Term Treatment. Stefano Menzo, 2004.The human immunodeficiency virus type 1 (HIV-1) fusion inhibitor enfuvirtide has recently been introduced into clinical practice and has exhibited efficient anti-HIV-1 activity in combination with other antiretroviral agents . In the present study, we addressed the effect of long-term treatment with enfuvirtide on the intrahost evolution of HIV-1 . The genotype and phenotype patterns and the relative replication capacity (rRC) of enfuvirtide-resistant HIV-1 mutants were evaluated in samples from 11 subjects (7 virological nonresponders and 4 responders) who received the compound for more than 1 year in combination with different regimens . Selection of one or more mutations clustering in a sequence (amino acids 36 to 45) of the gp41 N-terminal heptad repeat was observed in samples from the seven virological nonresponders but not in those from responders . In two subjects who discontinued enfuvirtide, reversion of the resistant genotype was detected within 3 months . Recombinant clones bearing mutated gp41 sequences displayed reduced susceptibilities to enfuvirtide, with the 50% inhibitory concentrations (IC50s) ranging from 0.6 to 12.8 µg/ml, whereas the IC50 for isolates with baseline sequences was 0.013 ± 0.010 µg/ml . Interestingly, long-term monitoring of resistant variants provided evidence that ongoing adaptation to the drug is paralleled by phenotypic changes . A limited drop in the rRC in the absence of drug was observed for clones from four of the seven nonresponders bearing mutations associated with resistance . Overall, the data indicate that the different genotype patterns associated with a detectable degree of HIV-1 resistance to enfuvirtide generated during long-term treatments are characterized by a substantially low genetic barrier, possible ongoing adaptation with increased degrees of resistance, and limited influence on the viral rRC . Chloromethane-Dependent Expression of the cmu Gene Cluster of Hyphomicrobium chloromethanicum. Elena Borodina, 2004.The methylotrophic bacterium Hyphomicrobium chloromethanicum CM2 can utilize chloromethane (CH3Cl) as the sole carbon and energy source . Previously genes cmuB, cmuC, cmuA, and folD were shown to be essential for the growth of Methylobacterium chloromethanicum on CH3Cl . These CH3Cl-specific genes were subsequently detected in H . chloromethanicum . Transposon and marker exchange mutagenesis studies were carried out to identify the genes essential for CH3Cl metabolism in H . chloromethanicum . New developments in genetic manipulation of Hyphomicrobium are presented in this study . An electroporation protocol has been optimized and successfully applied for transformation of mutagenesis plasmids into H . chloromethanicum to generate stable CH3Cl-negative mutants . Both transposon and marker exchange mutageneses were highly applicable for genetic analysis of Hyphomicrobium . A reliable and reproducible selection procedure for screening of CH3Cl utilization-negative mutants has also been developed . Mutational inactivation of cmuB, cmuC, or hutI resulted in strains that were unable to utilize CH3Cl or to express the CH3Cl-dependent polypeptide CmuA . Reverse transcription-PCR analysis indicated that cmuB, cmuC, cmuA, fmdB, paaE, hutI, and metF formed a single cmuBCA-metF operon and were coregulated and coexpressed in H . chloromethanicum . This finding led to the conclusion that, in cmuB and cmuC mutants, impaired expression of cmuA was likely to be due to a polar effect of the defective gene (cmuB or cmuC) located upstream (5') of cmuA . The detrimental effect of mutation in hutI on the upstream (5')-located cmuA is not clear but indicated that all the genes located within the cmuBCA-metF operon are coordinately expressed . Expression of the cmuBCA-metF transcript was also shown to be strictly CH3Cl inducible and was not repressed by the alternative C1 substrate methanol . Sequence analysis of a transposon mutant (D20) led to the discovery of the previously undetected hutI and metF genes located 3' of the paaE gene in H . chloromethanicum . MetF, a putative methylene-tetrahydrofolate reductase, had 27% identity to MetF from M . chloromethanicum . Mutational and transcriptional analysis data indicated that, in H . chloromethanicum, CH3Cl is metabolized via a corrinoid-specific (cmuA) and tetrahydrofolate-dependent (metF, purU, folD) methyltransfer system . Characterization of Two Tetrachloroethene-Reducing, Acetate-Oxidizing Anaerobic Bacteria and Their Description as Desulfuromonas michiganensis sp . nov.. Youlboong Sung, 2003.Two tetrachlorethene (PCE)-dechlorinating populations, designated strains BB1 and BRS1, were isolated from pristine river sediment and chloroethene-contaminated aquifer material, respectively . PCE-to-cis-1,2-dichloroethene-dechlorinating activity could be transferred in defined basal salts medium with acetate as the electron donor and PCE as the electron acceptor . Taxonomic analysis based on 16S rRNA gene sequencing placed both isolates within the Desulfuromonas cluster in the  subdivision of the Proteobacteria . PCE was dechlorinated at rates of at least 139 nmol min-1 mg of protein-1 at pH values between 7.0 and 7.5 and temperatures between 25 and 30°C . Dechlorination also occurred at 10°C . The electron donors that supported dechlorination included acetate, lactate, pyruvate, succinate, malate, and fumarate but not hydrogen, formate, ethanol, propionate, or sulfide . Growth occurred with malate or fumarate alone, whereas oxidation of the other electron donors depended strictly on the presence of fumarate, malate, ferric iron, sulfur, PCE, or TCE as an electron acceptor . Nitrate, sulfate, sulfite, thiosulfate, and other chlorinated compounds were not used as electron acceptors . Sulfite had a strong inhibitory effect on growth and dechlorination . Alternate electron acceptors (e.g., fumarate or ferric iron) did not inhibit PCE dechlorination and were consumed concomitantly . The putative fumarate, PCE, and ferric iron reductases were induced by their respective substrates and were not constitutively present . Sulfide was required for growth . Both strains tolerated high concentrations of PCE, and dechlorination occurred in the presence of free-phase PCE (dense non-aqueous-phase liquids) . Repeated growth with acetate and fumarate as substrates yielded a BB1 variant that had lost the ability to dechlorinate PCE . Due to the 16S rRNA gene sequence differences with the closest relatives and the unique phenotypic characteristics, we propose that the new isolates are members of a new species, Desulfuromonas michiganensis, within the Desulfuromonas cluster of the Geobacteraceae .
Conditional Survival as a Selection Strategy To Identify Plant-Inducible Genes of Pseudomonas syringae. Maria L. Marco, 2003.A novel strategy termed habitat-inducible rescue of survival (HIRS) was developed to identify genes of Pseudomonas syringae that are induced during growth on bean leaves . This strategy is based on the complementation of metXW, two cotranscribed genes that are necessary for methionine biosynthesis and required for survival of P . syringae on bean leaves exposed to conditions of low humidity . We constructed a promoter trap vector, pTrap, containing a promoterless version of the wild-type P . syringae metXW genes . Only with an active promoter fused to metXW on pTrap did this plasmid restore methionine prototrophy to the P . syringae metXW mutant B7MX89 and survival of this strain on bean leaves . To test this method, a partial library of P . syringae genomic DNA was constructed in pTrap and a total of 1,400 B7MX89 pTrap clones were subjected to HIRS selection on bean leaves . This resulted in the enrichment of five clones, each with a unique RsaI restriction pattern of their DNA insert . Sequence analysis of these clones revealed those P . syringae genes for which putative plant-inducible activity could be assigned . Promoter activity experiments with a gfp reporter gene revealed that these plant-inducible gene promoters had very low levels of expression in minimal medium . Based on green fluorescent protein fluorescence levels, it appears that many P . syringae genes have relatively low expression levels and that the metXW HIRS strategy is a sensitive method to detect weakly expressed P . syringae genes that are active on plants . Furthermore, we found that protected sites on the leaf surface provided a higher level of enrichment for P . syringae expressing metXW than exposed sites . Thus, the metXW HIRS strategy should lead to the identification of P . syringae genes that are expressed primarily in these areas on the leaf .
|
© 2005
Transgalactic Ltd (manufacturer of Bioscreen C software) |
Privacy Statement | P.O. Box
1393, 00101 Helsinki, Finland,
Last modified: May 25, 2005
| ||||||
