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Secretion of LamB-LacZ by the Signal Recognition Particle Pathway of Escherichia coli. Christina Wilson Bowers, 2003.LamB-LacZ fusion proteins have classically been used in studies of the general secretion pathway of Escherichia coli . Here we describe how increasing signal sequence hydrophobicity routes LamB-LacZ Hyb42-1 to the signal recognition particle (SRP) pathway . Secretion of this hydrophobic fusion variant (H*LamB-LacZ) was reduced in the absence of fully functional Ffh and Ffs, and the translocator jamming caused by Hyb42-1 was prevented by efficient delivery of the fusion to the periplasm . Finally, we found that in the absence of the ribosome-associated chaperone, trigger factor (Tig), LamB-LacZ localized to the periplasm in a SecA-dependent, SRP-independent fashion . Collectively, our results provide compelling in vivo evidence that there is an SRP-dependent cotranslational targeting mechanism in E . coli and argue against a role for trigger factor in pathway discrimination . Molecular Analysis of the Multiple GroEL Proteins of Chlamydiae. Karuna P. Karunakaran, 2003.Genome sequencing revealed that all six chlamydiae genomes contain three groEL-like genes (groEL1, groEL2, and groEL3) . Phylogenetic analysis of groEL1, groEL2, and groEL3 indicates that these genes are likely to have been present in chlamydiae since the beginning of the lineage . Comparison of deduced amino acid sequences of the three groEL genes with those of other organisms showed high homology only for groEL1, although comparison of critical amino acid residues that are required for polypeptide binding of the Escherichia coli chaperonin GroEL revealed substantial conservation in all three chlamydial GroELs . This was further supported by three-dimensional structural predictions . All three genes are expressed constitutively throughout the developmental cycle of Chlamydia trachomatis, although groEL1 is expressed at much higher levels than are groEL2 and groEL3 . Transcription of groEL1, but not groEL2 and groEL3, was elevated when HeLa cells infected with C . trachomatis were subjected to heat shock . Western blot analysis with polyclonal antibodies raised against recombinant GroEL1, GroEL2, and GroEL3 demonstrated the presence of the three proteins in C . trachomatis elementary bodies, with GroEL1 being present in the largest amount . Only C . trachomatis groEL1 and groES together complemented a temperature-sensitive E . coli groEL mutant . Complementation did not occur with groEL2 or groEL3 alone or together with groES . The role for each of the three GroELs in the chlamydial developmental cycle and in disease pathogenesis requires further study . Large-Restriction-Fragment Polymorphism Analysis of Mycobacterium chelonae and Mycobacterium terrae Isolates. J. Daisy Vanitha, 2003.Mycobacterium chelonae and Mycobacterium terrae were reported to be frequently present in the environment of the Mycobacterium bovis BCG trial area in south India . Six isolates of M . chelonae and four isolates of M . terrae obtained from different sources in this area were analyzed by pulsed-field gel electrophoresis (PFGE) to examine large-restriction-fragment (LRF) polymorphism using the chromosomal DNA digested with DraI and XbaI restriction enzymes . With the exception of one isolate of M . terrae, DNA from all other isolates could be digested with DraI and XbaI and resulted in separable fragments . Visual comparison of the LRFs showed a unique pattern for each of the isolates tested . A computer-assisted dendrogram of the percent similarity demonstrated a high degree of genetic diversity in this group of isolates . This study demonstrates that species of nontuberculous mycobacteria, particularly M . chelonae and M . terrae, can be successfully typed by their LRF pattern using PFGE, which does not require species-specific DNA probes .
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