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Evolution of a Pathway to Novel Long-Chain Carotenoids. Daisuke Umeno, 2004.Using methods of laboratory evolution to force the C30 carotenoid synthase CrtM to function as a C40 synthase, followed by furthermutagenesis at functionally important amino acid residues, we have discovered that synthase specificity is controlled at the second [rearrangement] step of the two-step reaction . We usedthis information to engineer CrtM variants that can synthesizepreviously unknown C45 and C50 carotenoid backbones [mono- and diisopentenylphytoenes] from the appropriate isoprenyldiphosphate precursors . With this ability to produce new backbones in Escherichia coli comes the potential to generate whole series of novel carotenoids by using carotenoid-modifying enzymes, including desaturases, cyclases, hydroxylases, and dioxygenases, from naturally occurring pathways. Comparison of the Susceptibilities of Burkholderia pseudomallei to Meropenem and Ceftazidime by Conventional and Intracellular Methods. T. J. J. Inglis, 2004.The effect of the two antibiotics ceftazidime and meropenem on a collection of 46 Burkholderia pseudomallei isolates representing clinical and environmental sources across northern Australia was investigated by using a series of in vitro test methods . The susceptibility testing methods used included Kirby-Bauer disk diffusion, Etest MIC, broth microdilution MIC, and a modification of the microdilution method in which Acanthamoeba cells were added to simulate the effect of a professional phagocytic cell on test outcome . In a semiquantitative validation coculture series, the majority of bacteria were intracellular up to a multiplicity of infection of 10 bacteria to one ameba . The optical density and bacterial count (log10 CFU/ml) correlated across the range tested (r2 = 0.77; P < 0.0001) . Susceptibility test results were compared against clinical outcomes . The MICs of ceftazidime were consistently higher than those of meropenem by all three methods . The MICs of both agents were significantly higher when Acanthamoeba trophozoites were added to the broth microdilution method . Conventional and intracellular MIC results were consistent for clinical isolates from the Western Australian outbreak cluster despite the wide variety of clinical outcomes . Further development of the intracellular MIC method is expected to help assess the efficacy of antimicrobial agents on this bacterial species in an intracellular setting . Chlorobium tepidum Mutant Lacking Bacteriochlorophyll c Made by Inactivation of the bchK Gene, Encoding Bacteriochlorophyll c Synthase. Niels-Ulrik Frigaard, 2002.The gene encoding bacteriochlorophyll (BChl) c synthase was identified by insertional inactivation in the photosynthetic green sulfur bacterium Chlorobium tepidum and was named bchK . The bchK mutant of C . tepidum was rusty-orange in color and completely lacked BChl c . Because of the absence of the BChl c antenna, the mutant grew about seven times slower than the wild type at light intensities that were limiting to the wild type (<90 µmol m-2 s-1) . Various pheophorbides, which probably represent precursors of BChl c which had lost magnesium, accumulated in the mutant cells . A small fraction of these pheophorbides were apparently esterified by the remaining chlorophyll (Chl) a and BChl a synthases in cells . The amounts of BChl a, Chl a, isoprenoid quinones, carotenoids, Fenna-Matthews-Olson protein, and chlorosome envelope protein CsmA were not significantly altered on a cellular basis in the mutant compared to in the wild type . This suggests that the BChl a antennae, photosynthetic reaction centers, and remaining chlorosome components were essentially unaffected in the mutant . Electron microscopy of thin sections revealed that the mutant lacked normal chlorosomes . However, a fraction containing vestigial chlorosomes, denoted "carotenosomes," was partly purified by density centrifugation; these structures contained carotenoids, isoprenoid quinones, and a 798-nm-absorbing BChl a species that is probably protein associated . Because of the absence of the strong BChl c absorption found in the wild type, the bchK mutant should prove valuable for future analyses of the photosynthetic reaction center and of the roles of BChl a in photosynthesis in green bacteria . An evolutionary implication of our findings is that the photosynthetic ancestor of green sulfur bacteria could have evolved without chlorosomes and BChl c and instead used only BChl a-containing proteins as the major light-harvesting antennae . Regulation of the Bacillus subtilis bcrC Bacitracin Resistance Gene by Two Extracytoplasmic Function  Factors.Min Cao, 2002.Bacitracin resistance is normally conferred by either of two major mechanisms, the BcrABC transporter, which pumps out bacitracin, or BacA, an undecaprenol kinase that provides C55-isoprenyl phosphate by de novo synthesis . We demonstrate that the Bacillus subtilis bcrC (ywoA) gene, encoding a putative bacitracin transport permease, is an important bacitracin resistance determinant . A bcrC mutant strain had an eightfold-higher sensitivity to bacitracin . Expression of bcrC initiated from a single promoter site that could be recognized by either of two extracytoplasmic function (ECF) factors,
X or
M . Bacitracin induced expression of bcrC, and this induction was dependent on
M but not on
X . Under inducing conditions, expression was primarily dependent on
M . As a consequence, a sigM mutant was fourfold more sensitive to bacitracin, while the sigX mutant was only slightly sensitive . A sigX sigM double mutant was similar to a bcrC mutant in sensitivity . These results support the suggestion that one function of B . subtilis ECF
factors is to coordinate antibiotic stress responses .
Transcriptional Regulation and Signature Patterns Revealed by Microarray Analyses of Streptococcus pneumoniae R6 Challenged with Sublethal Concentrations of Translation Inhibitors. Wai-Leung Ng, 2003.The effects of sublethal concentrations of four different classes of translation inhibitors (puromycin, tetracycline, chloramphenicol, and erythromycin) on global transcription patterns of Streptococcus pneumoniae R6 were determined by microarray analyses . Consistent with the general mode of action of these inhibitors, relative transcript levels of genes that encode ribosomal proteins and translation factors or that mediate tRNA charging and amino acid biosynthesis increased or decreased, respectively . Transcription of the heat shock regulon was induced only by puromycin or streptomycin treatment, which lead to truncation or mistranslation, respectively, but not by other antibiotics that block translation, transcription, or amino acid charging of tRNA . In contrast, relative transcript amounts of certain genes involved in transport, cellular processes, energy metabolism, and purine nucleotide (pur) biosynthesis were changed by different translation inhibitors . In particular, transcript amounts from a pur gene cluster and from purine uptake and salvage genes were significantly elevated by several translation inhibitors, but not by antibiotics that target other cellular processes . Northern blotting confirmed increased transcript amounts from part of the pur gene cluster in cells challenged by translation inhibitors and revealed the presence of a 10-kb transcript . Purine metabolism genes were negatively regulated by a homologue of the PurR regulatory protein, and full derepression in a  purR mutant depended on optimal translation . Unexpectedly, hierarchical clustering of the microarray data distinguished among the global transcription patterns caused by antibiotics that inhibit different steps in the translation cycle . Together, these results show that there is extensive control of transcript amounts by translation in S . pneumoniae, especially for de novo purine nucleotide biosynthesis . In addition, these global transcription patterns form a signature that can be used to classify the mode of action and potential mechanism of new translation inhibitors .
Precise Excision of the Large Pathogenicity Island, SPI7, in Salmonella enterica Serovar Typhi. Susan M. Bueno, 2004.
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