Ethanol-induced stress response of Staphylococcus aureus.

Transcriptional profiles of 2 unrelated clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates were analyzed following 10% (v/v) ethanol challenge (15 min), which arrested growth but did not reduce viability. Ethanol-induced stress (EIS) resulted in differential gene expression of 1091 genes, 600 common to both strains, of which 291 were upregulated. With the exception of the downregulation of genes involved with osmotic stress functions, EIS resulted in the upregulation of genes that contribute to stress response networks, notably those altered by oxidative stress, protein quality control in general, and heat shock in particular. In addition, genes involved with transcription, translation, and nucleotide biosynthesis were downregulated. relP, which encodes a small alarmone synthetase (RelP), was highly upregulated in both MRSA strains following ethanol challenge, and relP inactivation experiments indicated that this gene contributed to EIS growth arrest. A number of persistence-associated genes were also upregulated during EIS, including those that encode toxin-antitoxin systems. Overall, transcriptional profiling indicated that the MRSA investigated responded to EIS by entering a state of dormancy and by altering the expression of elements from cross protective stress response systems in an effort to protect preexisting proteins.

Gene encoding gamma-carbonic anhydrase is cotranscribed with argC and induced in response to stationary phase and high CO2 in Azospirillum brasilense Sp7.

BACKGROUND: Carbonic anhydrase (CA) is a ubiquitous enzyme catalyzing the reversible hydration of CO2 to bicarbonate, a reaction underlying diverse biochemical and physiological processes. Gamma class carbonic anhydrases (gamma-CAs) are widespread in prokaryotes but their physiological roles remain elusive. At present, only gamma-CA of Methanosarcina thermophila (Cam) has been shown to have CA activity. Genome analysis of a rhizobacterium Azospirillum brasilense, revealed occurrence of ORFs encoding one beta-CA and two gamma-CAs. RESULTS: One of the putative gamma-CA encoding genes of A. brasilense was cloned and overexpressed in E. coli. Electrometric assays for CA activity of the whole cell extracts overexpressing recombinant GCA1 did not show CO2 hydration activity. Reverse transcription-PCR analysis indicated that gca1 in A. brasilense is co-transcribed with its upstream gene annotated as argC, which encodes a putative N-acetyl-gamma-glutamate-phosphate reductase. 5'-RACE also demonstrated that there was no transcription start site between argC and gca1, and the transcription start site located upstream of argC transcribed both the genes (argC-gca1). Using transcriptional fusions of argC-gca1 upstream region with promoterless lacZ, we further demonstrated that gca1 upstream region did not have any promoter and its transcription occurred from a promoter located in the argC upstream region. The transcription of argC-gca1 operon was upregulated in stationary phase and at elevated CO2 atmosphere. CONCLUSIONS: This study shows lack of CO2 hydration activity in a recombinant protein expressed from a gene predicted to encode a gamma-carbonic anhydrase in A. brasilense although it cross reacts with anti-Cam antibody raised against a well characterized gamma-CA. The organization and regulation of this gene along with the putative argC gene suggests its involvement in arginine biosynthetic pathway instead of the predicted CO2 hydration.

Transcriptional targeting of human liver carboxylesterase (hCE1m6) and simultaneous expression of anti-BCRP shRNA enhances sensitivity of breast cancer cells to CPT-11.

BACKGROUND: The major factor limiting the efficacy of breast cancer chemotherapy is multidrug resistance due to overexpression of the breast cancer resistance protein ATP-binding cassette, sub-family G (WHITE), member 2 (ABCG2). We hypothesized that conversion of camptothecin-11 (CPT-11) to its highly cytotoxic metabolite SN-38 by a mutant human carboxyl esterase (hCE1m6) specifically in cancer cells and inhibition of ABCG2 by anti-ABCG2 short hairpin RNA, leads to accumulation of a higher concentration of SN-38, resulting in higher therapeutic efficacy and less toxicity to normal cells. MATERIALS AND METHODS: A mutant human carboxyl esterase hCE1m6 with human telomerase reverse transcriptase promoter was integrated into the VISA (VP16-Gal4-WPRE) amplification system. The plasmid was transfected into MCF-12A, MDA-MB-231, and MCF-7 cells using JetPRIME®. Cancer-specific expression of hCE1m6 in breast cancer cell lines was tested by real-time polymerase chain reaction (real time-PCR) and western blot. In vitro conversion of CPT-11 to SN-38 was evaluated on lysates of transfected cells. Cytotoxicity of CPT-11 against cells transfected with the plasmid was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS: Real-time PCR and western blot analysis revealed that hCE1m6 was expressed only in breast cancer cells, MCF-7 and MDA-MB-231, but not in the normal MCF-12A breast cell line. From the CPT-11 conversion assay on cell lysates, it was found that expressed hCE1m6 in cancer cells was able to effectively convert CPT-11 to SN-38. CONCLUSION: Breast cancer cell lines transfected with hCE1m6 showed an increased susceptibility to CPT-11 in comparison to MCF-12A cells.