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Construction and Validation of the Rhodobacter sphaeroides 2.4.1 DNA Microarray: Transcriptome Flexibility at Diverse Growth Modes. Christopher T. Pappas, 2004.A high-density oligonucleotide DNA microarray, a genechip, representing the 4.6-Mb genome of the facultative phototrophic proteobacterium, Rhodobacter sphaeroides 2.4.1, was custom-designed and manufactured by Affymetrix, Santa Clara, Calif . The genechip contains probe sets for 4,292 open reading frames (ORFs), 47 rRNA and tRNA genes, and 394 intergenic regions . The probe set sequences were derived from the genome annotation generated by Oak Ridge National Laboratory after extensive revision, which was based primarily upon codon usage characteristic of this GC-rich bacterium . As a result of the revision, numerous missing ORFs were uncovered, nonexistent ORFs were deleted, and misidentified start codons were corrected . To evaluate R . sphaeroides transcriptome flexibility, expression profiles for three diverse growth modes—aerobic respiration, anaerobic respiration in the dark, and anaerobic photosynthesis—were generated . Expression levels of one-fifth to one-third of the R . sphaeroides ORFs were significantly different in cells under any two growth modes . Pathways involved in energy generation and redox balance maintenance under three growth modes were reconstructed . Expression patterns of genes involved in these pathways mirrored known functional changes, suggesting that massive changes in gene expression are the major means used by R . sphaeroides in adaptation to diverse conditions . Differential expression was observed for genes encoding putative new participants in these pathways (additional photosystem genes, duplicate NADH dehydrogenase, ATP synthases), whose functionality has yet to be investigated . The DNA microarray data correlated well with data derived from quantitative reverse transcription-PCR, as well as with data from the literature, thus validating the R . sphaeroides genechip as a powerful and reliable tool for studying unprecedented metabolic versatility of this bacterium . Comparative Sequence Analysis of the Symbiosis Island of Mesorhizobium loti Strain R7A. John T. Sullivan, 2002.The Mesorhizobium loti strain R7A symbiosis island is a 502-kb chromosomally integrated element which transfers to nonsymbiotic mesorhizobia in the environment, converting them to Lotus symbionts . It integrates into a phenylalanine tRNA gene in a process mediated by a P4-type integrase encoded at the left end of the element . We have determined the nucleotide sequence of the island and compared its deduced genetic complement with that reported for the 611-kb putative symbiosis island of M . loti strain MAFF303099 . The two islands share 248 kb of DNA, with multiple deletions and insertions of up to 168 kb interrupting highly conserved colinear DNA regions in the two strains . The shared DNA regions contain all the genes likely to be required for Nod factor synthesis, nitrogen fixation, and island transfer . Transfer genes include a trb operon and a cluster of potential tra genes which are also present on the strain MAFF303099 plasmid pMLb . The island lacks plasmid replication genes, suggesting that it is a site-specific conjugative transposon . The R7A island encodes a type IV secretion system with strong similarity to the vir pilus from Agrobacterium tumefaciens that is deleted from MAFF303099, which in turn encodes a type III secretion system not found on the R7A island . The 414 genes on the R7A island also include putative regulatory genes, transport genes, and an array of metabolic genes . Most of the unique hypothetical genes on the R7A island are strain-specific and clustered, suggesting that they may represent other acquired genetic elements rather than symbiotically relevant DNA . Yersinia enterocolitica Type III Secretion: yscM1 and yscM2 Regulate yop Gene Expression by a Posttranscriptional Mechanism That Targets the 5' Untranslated Region of yop mRNA. Eric D. Cambronne, 2002. Heat Adaptation Alters Escherichia coli O157:H7 Membrane Lipid Composition and Verotoxin Production. Hyun-Gyun Yuk, 2003.The influence of heat adaptation (growth at 42 and 45°C) on changes in membrane lipid composition and verotoxin concentration of Escherichia coli O157:H7 (ATCC 43895), an rpoS mutant of ATCC 43895 (FRIK 816-3), a verotoxin mutant E . coli O157:H7 (B6-914), and nonpathogenic E . coli (ATCC 25922) was investigated . D values (57°C) of heat-adapted cells were up to 3.9 min longer than those of control cells for all four strains . Heat adaptation increased the amounts of palmitic acid (16:0) and cis-vaccenic acid (18:1  7c) in membrane lipids of ATCC 43895 and the rpoS mutant, whereas there was a reduction and no change in the amount of cis-vaccenic acid in nonpathogenic and verotoxin mutant E . coli, respectively . The ratio of palmitic to cis-vaccenic acids decreased in ATCC 43895 and in the rpoS mutant, whereas the ratio increased in nonpathogenic E . coli and was not different in the verotoxin mutant with elevated growth temperature . Total verotoxin concentration decreased due to a reduction in intracellular verotoxin amount in heat-adapted ATCC 43895 and rpoS mutant strains . However, extracellular verotoxin concentration increased in heat-adapted cells . The rpoS gene did not influence membrane lipid composition changes although it did affect heat resistance . Results suggest that increased membrane fluidity may have caused increased verotoxin secretion .
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