1743Many aromatic compounds are toxic when supplied at concentrations employed in most growth media. This effect was demonstrated when rhizobia and agrobacteria were grown in auxanographic plates in which cells were seeded in agar and exposed to a gentle gradient of substrate concentration. An auxanographic nutritional survey with representative strains revealed that Rhizobium japonicum and cowpea Rhizobium sp. could utilize a relatively large proportion of the aromatic and hydroaromatic compounds tested; Rhizobiurn legurninosarum, Rhizobium trifolii and Agrobacterium species displayed intermediate nutritional versatility ; Rhizobium meliloti was relatively fastidious. The hydroaromatics quinate and shikimate were not toxic. Most of the strains examined grew at the expense of one or both of these substrates. Quinate was metabolized via protocatechuate and 3-oxoadipate. All of the strains examined were able to grow at the expense of protocatechuate and therefore must contain six structural genes for the enzymes required to convert this aromatic compound to common intermediary metabolites. Conservation of pathways for aromatic catabolism against a background of wide evolutionary divergence among the Rhizobiaceae suggests that pressures for selection of the traits were exerted throughout the evolutionary history of the organisms. A probable selective pressure is competition for nutrients in the soil. In addition, the ability of agrobacteria and rhizobia to respond to aromatic compounds may have selective value in bacterial-plant interactions.

The genetic and physiological properties of Acinetobacter baylyi strain ADP1 make it an inviting subject for investigation of the properties underlying its nutritional versatility. The organism possesses a relatively small genome in which genes for most catabolic functions are clustered in several genetic islands that, unlike pathogenicity islands, give little evidence of horizontal transfer. Coupling mutagenic polymerase chain reaction to natural transformation provides insight into how structure influences function in transporters, transcriptional regulators, and enzymes. With appropriate selection, mutants in which such molecules have acquired novel function may be obtained. The extraordinary competence of A. baylyi for natural transformation and the ease with which it expresses heterologous genes make it a promising platform for construction of novel metabolic systems. Steps toward this goal should take into account the complexity of existing pathways in which transmembrane trafficking plays a significant role.

Berberine [Natural Yellow 18, 5,6-dihydro-9,10-dimethoxybenzo(g)-1,3-benzodioxolo(5,6-a)quinolizinium] is an alkaloid present in plant extracts and has a history of use in traditional Chinese and Native American medicine. Because of its ability to arrest the cell cycle and cause apoptosis of several malignant cell lines, it has received attention as a potential anticancer therapeutic agent. Previous studies suggest that mitochondria may be an important target of berberine, but relatively little is known about the extent or molecular mechanisms of berberine-mitochondrial interactions. The objective of the present work was to investigate the interaction of berberine with mitochondria, both in situ and in isolated mitochondrial fractions. The data show that berberine is selectively accumulated by mitochondria, which is accompanied by arrest of cell proliferation, mitochondrial fragmentation and depolarization, oxidative stress, and a decrease in ATP levels. Electron microscopy of berberine-treated cells shows a reduction in mitochondria-like structures, accompanied by a decrease in mitochondrial DNA copy number. Isolated mitochondrial fractions treated with berberine had slower mitochondrial respiration, especially when complex I substrates were used, and increased complex I-dependent oxidative stress. It is also demonstrated for the first time that berberine stimulates the mitochondrial permeability transition. Direct effects on ATPase activity were not detected. The present work demonstrates a number of previously unknown alterations of mitochondrial physiology induced by berberine, a potential chemotherapeutic agent, although it also suggests that high doses of berberine should not be used without a proper toxicology assessment.Berberine (Fig.

Purpose Natural products represent a rich reservoir of potential small molecule inhibitors exhibiting antiproliferative and tumoricidal properties. An example is the isoquinoline alkaloid berberine, which is found in plants such as goldenseal (Hydrastis canadensis). Studies have shown that berberine is able to trigger apoptosis in diVerent malignant cell lines, and can also lead to cell cycle arrest at sub-apoptotic doses. A particularly interesting feature of berberine is the fact that it is a Xuorescent molecule, and its uptake and distribution in cells can be studied by Xow cytometry and epiXuorescence microscopy. To test the relationships between berberine uptake, distribution and cellular eVect in melanoma cells, K1735-M2 mouse and WM793 human melanoma cells were treated with diVerent concentrations of berberine, and alterations in cell cycle progression, DNA synthesis, cell proliferation, and cell death measured. Methods Cell proliferation was measured by sulforhodamine B assays, cell death by Xow cytometry, berberine uptake and distribution by laser scanning confocal microscopy and Xow cytometry, cell cycle progression by Xow cytometry, and DNA synthesis, M-phase, and mitochondrial eVects by immunolabeling and epiXuorescence microscopy methods. Results In these melanoma cell lines, berberine at low doses (12.5-50 M) is concentrated in mitochondria and promotes G1 arrest. In contrast, higher doses (over 50 M) result in cytoplasmic and nuclear berberine accumulation, and G2 arrest. DNA synthesis is not markedly aVected by low doses of berberine, but 100 M is strongly inhibitory. Even at 100 M, berberine inhibits cell growth with relatively little induction of apoptosis. Conclusion Berberine displays multiphasic eVects in these malignant cell lines, which are correlated with the concentration and intracellular distribution of this alkaloid. These results help explain some of the conXicting information in the literature regarding the eVects of berberine, and suggest that its use in clinical development may be more as a cytostatic agent than a cytotoxic compound.

Hydroxycinnamates are plant products catabolized through the diphenol protocatechuate in the naturally transformable bacterium Acinetobacter sp. strain ADP1. Genes for protocatechuate catabolism are central to the dca-pca-qui-pob-hca chromosomal island, for which gene designations corresponding to catabolic function are dca (dicarboxylic acid), pca (protocatechuate), qui (quinate), pob (p-hydroxybenzoate), and hca (hydroxycinnamate). Acinetobacter hcaC had been cloned and shown to encode a hydroxycinnamate:coenzyme A (CoA) SH ligase that acts upon caffeate, p-coumarate, and ferulate, but genes for conversion of hydroxycinnamoylCoA to protocatechuate had not been characterized. In this investigation, DNA from pobS to an XbaI site 5.3 kb beyond hcaC was captured in the plasmid pZR8200 by a strategy that involved in vivo integration of a cloning vector near the hca region of the chromosome. pZR8200 enabled Escherichia coli to convert p-coumarate to protocatechuate in vivo. Sequence analysis of the newly cloned DNA identified five open reading frames designated hcaA, hcaB, hcaK, hcaR, and ORF1. An Acinetobacter strain with a knockout of HcaA, a homolog of hydroxycinnamoyl-CoA hydratase/lyases, was unable to grow at the expense of hydroxycinnamates, whereas a strain mutated in HcaB, homologous to aldehyde dehydrogenases, grew poorly with ferulate and caffeate but well with p-coumarate. A chromosomal fusion of lacZ to the hcaE gene was used to monitor expression of the hcaABCDE promoter. LacZ was induced over 100-fold by growth in the presence of caffeate, p-coumarate, or ferulate. The protein deduced to be encoded by hcaR shares 28% identity with the aligned E. coli repressor, MarR. A knockout of hcaR produced a constitutive phenotype, as assessed in the hcaE::lacZ-Km r genetic background, revealing HcaR to be a repressor as well. Expression of hcaE::lacZ in strains with knockouts in hcaA, hcaB, or hcaC revealed unambiguously that hydroxycinnamoyl-CoA thioesters relieve repression of the hcaABCDE genes by HcaR.