A cerulenin insensitive 3-ketoacyl-acyl carrier protein synthase has been assayed in extracts of spinach (Spinacia oleracea) leaf. The enzyme was active in the 40 to 80% ammonium sulfate precipitate of whole leaf homogenates and catalyzed the synthesis of acetoacetyl-acyl carrier protein. This condensation reaction was five-fold faster than acetyl-CoA:acyl carrier protein transacylase, and the initial rates of acyl-acyl carrier protein synthesis were independent of the presence of cerulenin. In the presence of fatty acid synthase cofactors and 100 micromolar cerulenin, the principal fatty acid product of de novo synthesis was butyric and hexanoic acids. Using conformationally sensitive native polyacrylamide gel electrophoresis for separation, malonyl-, acetyl-, butyryl-, hexanoyl, and long chain acyl-acyl carrier proteins could be detected by immunoblotting and autoradiography. In the presence of 100 micromolar cerulenin, the accumulation of butyryland hexanoyl-acyl carrier protein was observed, with no detectable long chain acyl-acyl carrier proteins or fatty acids being produced. In the absence of cerulenin, the long chain acyl-acyl carrier proteins also accumulated.biosynthesis to cerulenin can be attributed entirely to the inhibition of 3-ketoacyl-ACP synthase I.Similarly, the 3-ketoacyl-ACP synthases found in Escherichia coli, are characteristically cerulenin sensitive (5). Recently, a third condensing enzyme has been reported in E. coli. which has been named acetoacetyl-ACP synthase (6). This 3-ketoacyl-ACP synthase is distinctly different from the other 3-ketoacyl-ACP synthases in several important respects. It is cerulenin insensitive, is specific for very short chain acylACPs, and it prefers acetyl-CoA over acetyl-ACP (7). As a consequence of this last property, it would appear that this enzyme can bypass the much slower acetyl transacylase step and as a result, the acetyl transacylase would not be rate limiting in fatty acid biosynthesis.In light of these findings in the E. coli system, we have reexamined the spinach system and report here the presence ofa third 3-ketoacyl-ACP synthase which is completely insensitive to cerulenin, active only with short chain acyl thioesters, and appears to prefer acetyl-CoA to acetyl-ACP. MATERIALS AND METHODS MaterialsIt has been known for decades that fatty acid biosynthesis involves extending an acyl-ACP' stepwise by two carbons. In plants and bacteria, this synthesis is catalyzed by a type II fatty acid synthase, a readily dissociable group of enzymes which can be individually isolated and studied (1). One of the initial reactions leading to chain elongation is catalyzed by the enzyme 3-ketoacyl-ACP synthase, frequently referred to simply as the 'condensing enzyme.' The spinach fatty acid synthase was initially separated into its individual components by Shimakata and Stumpf (13,14). In that study, they found two 3-ketoacyl-ACP synthases, designated I and II. 3-Ketoacyl-ACP synthase I was responsible for the majority of the condensations, using acyl-ACPs r...
The low molecular weight (LMW) heat shock protein (HSP), HSP16.6, in the unicellular cyanobacterium, Synechocystis sp. PCC 6803, protects cells from elevated temperatures. A 95% reduction in the survival of mutant cells with an inactivated hsp16.6 was observed after exposure for 1 h at 47 degrees C. Wild-type cell survival was reduced to only 41%. HSP16.6 is also involved in the development of thermotolerance. After a sublethal heat shock at 43 degrees C for 1 h and subsequent challenge exposure at 49 degrees C for 40 min, mutant cells did not survive, while 64% of wild-type cells survived. Ultrastructural changes in the integrity of thylakoid membranes of heat-shocked mutant cells also are discussed. These results demonstrate an important protective role for HSP16.6 in the protection of cells and, in particular, thylakoid membrane against thermal stress.
The low molecular weight (LMW) heat shock protein (HSP) gene hsp16.6 was identified and cloned from the unicellular cyanobacterium Synechocystis sp. PCC 6803 through comparisons of genomic sequences and conserved gene sequences of the LMW HSPs. Hsp16.6 was isolated using PCR and cloned into the pGEMT plasmid. Hsp16.6 showed a significant increase in transcription after heat shock at 42 degreesC that indicated hsp16.6 was a heat shock gene. To determine the role that hsp16.6 plays in the heat shock response, a mutant Synechocystis cell line was generated. Cell growth and oxygen evolution rates of wild type and mutant cells were compared after heat shock. Results showed significantly decreased cell growth rates and a 40% reduction in oxygen evolution rates in mutants after heat shock treatments. These data indicate a protective role for hsp16.6 in the heat shock response.
The heterocystous cyanobacteria are currently placed in subsections IV and V, which are distinguished by cellular division in one plane (false branching) and in more than one plane (true branching), respectively. Published phylogenies of 16S rRNA gene sequence data support the monophyly of the heterocystous cyanobacteria, with members of subsection V embedded within subsection IV. It has been postulated that members of subsection V arose from within subsection IV. Therefore, phylogenetic analysis of nucleotide sequences of the nitrogen-fixation gene nifD from representatives of subsections IV and V was performed by using maximum-likelihood criteria. The heterocystous cyanobacteria are supported as being monophyletic, with the non-heterocystous cyanobacteria as their closest relative. However, neither subsection IV nor subsection V is monophyletic, with representatives of both subsections intermixed in two sister clades. Analysis of nifD does not support recognition of two distinct subsections.Cyanobacteria are oxygenic, photosynthetic prokaryotes that can be found in almost every aquatic and terrestrial environment (Castenholz & Waterbury, 1989) and are ancient organisms that date back 3?5 billion years in the fossil record (Castenholz, 1992). It has been proposed that cyanobacteria were responsible for converting the ancient Earth's anaerobic atmosphere to an aerobic one (Hayes, 1983;Schopf et al., 1983). Many cyanobacteria fix nitrogen (diazotrophy) and they comprise one of the largest global suppliers of fixed nitrogen in the environment (Sprent & Sprent, 1990). In addition, some species of nitrogen-fixing cyanobacteria are involved in symbiotic relationships with many plant species and supply the host directly with a source of reduced nitrogen.The taxonomy of the cyanobacteria has been debated vigorously and revised many times (Golubic, 1976;Turner, 1997). The cyanobacteria have been reclassified by using bacteriological instead of botanical criteria, based on morphological and developmental features, but most species, including those used in this study, have not yet been given validly published names under the Bacteriological Code. Cyanobacteria are currently divided into five subsections (Rippka et al., 1979;Rippka, 1988;Castenholz & Waterbury, 1989;Castenholz, 1992Castenholz, , 2001Rippka & Herdman, 1992). Subsection I strains are unicellular and divide by binary fission or budding (Rippka et al., 1979). Subsection II strains are also unicellular but divide by multiple fission, resulting in the formation of baeocytes (Rippka et al., 1979;Rippka, 1988;Castenholz & Waterbury, 1989;Castenholz, 1992Castenholz, , 2001Rippka & Herdman, 1992). Subsection III strains are filamentous, non-heterocystous cyanobacteria that reproduce by trichome breakage (Rippka et al., 1979;Rippka, 1988;Castenholz & Waterbury, 1989;Castenholz, 1992Castenholz, , 2001Rippka & Herdman, 1992). Subsections IV and V are composed exclusively of heterocystous cyanobacteria, which are filamentous strains that reproduce by hormogonia formatio...
The ability to fix nitrogen is widely, but sporadically distributed among the Bacteria and Archaea suggesting either a vertically inherited, ancient function with widespread loss across genera or an adaptive feature transferred laterally between co-inhabitants of nitrogen-poor environments. As previous phylogenetic studies of nifH and nifD have not completely resolved the evolutionary history of nitrogenase, sixty nifD, nifK, and combined nifDK genes were analyzed using Bayesian, maximum likelihood, and parsimony algorithms to determine whether the individual and combined datasets could provide additional information. The results show congruence between the 16S and nifDK phylogenies at the phyla level and generally support vertical descent with loss. However, statistically significant differences between tree topographies suggest a complex evolutionary history with the underlying pattern of vertical descent obscured by recurring lateral transfer events and different patterns of evolution between the genes. Results support inheritance from the Last Common ancestor or an ancient lateral transfer of the nif genes between Bacteria and Archaea, ongoing gene transfer between cohabitants of similar biogeographic regions, acquisition of nitrogen-fixing capability via symbiosis islands, possible xenologous displacement of one gene in the operon, and possible retention of ancestral genes in heterocystous cyanobacteria. Analyses support the monophyly of the Cyanobacteria, alphabetagamma-Proteobacteria, and Actinobacteria (Frankia) and provide strong support for the placement of Frankia nif genes at the base of combined the Cyanobacteria/Proteobacteria clades.
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