Oxygen-dependent autoaggregation in Shewanella oneidensis MR-1.

In aerobic chemostat cultures maintained at 50% dissolved O(2) tension (3.5 mg l(-1) dissolved O(2)), Shewanella oneidensis strain MR-1 rapidly aggregated upon addition of 0.68 mM CaCl(2) and retained this multicellular phenotype at high dilution rates. Confocal microscopy analysis of the extracellular matrix material contributing to the stability of the aggregate structures revealed the presence of extracellular DNA, protein and glycoconjugates. Upon onset of O(2)-limited growth (dissolved O(2) below detection) however, the Ca(2+)-supplemented chemostat cultures of strain MR-1 rapidly disaggregated and grew as motile dispersed cells. Global transcriptome analysis comparing aerobic aggregated to O(2)-limited unaggregated cells identified genes encoding cell-to-cell and cell-to-surface adhesion factors whose transcription increased upon exposure to increased O(2) concentrations. The aerobic aggregated cells also revealed increased expression of putative anaerobic electron transfer and homologues of metal reduction genes, including mtrD (SO1782), mtrE (SO1781) and mtrF (SO1780). Our data indicate that mechanisms involved in autoaggregation of MR-1 are dependent on the function of pilD gene which encodes a putative prepilin peptidase. Mutants of S. oneidensis strain MR-1 deficient in PilD and associated pathways, including type IV and Msh pili biogenesis, displayed a moderate increase in sensitivity to H(2)O(2). Taken together, our evidence indicates that aggregate formation in S. oneidensis MR-1 may serve as an alternative or an addition to biochemical detoxification to reduce the oxidative stress associated with production of reactive oxygen species during aerobic metabolism while facilitating the development of hypoxic conditions within the aggregate interior.

Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR.

The effects of serum on the expression of four commonly used housekeeping genes were examined in serum-stimulated fibroblasts in order to validate the internal control genes for a quantitative RT-PCR assay. NIH 3T3 fibroblasts transfected with an inducible chimeric gene were serum-starved for 24 h and then induced with 15% serum for 8 h. Serum did not alter the amount of total RNA that was expressed in the cells, however, the amount of mRNA significantly increased over time with serum-stimulation. Both messenger and total RNA from each of the time points were reverse transcribed under two different conditions; one in which the reactions were normalized to contain equal amounts of RNA and another series of reactions that were not normalized to RNA content. The resulting cDNA was amplified by real-time, quantitative PCR using gene-specific primers for beta-actin, beta-2 microglobulin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 18S ribosomal RNA. The expression of beta-actin and GAPDH increased up to nine- and three-fold, respectively, under all conditions of reverse transcription (P<0.01). The expression of 18S rRNA increased with serum-stimulation when the cDNA synthesized from non-normalized, total RNA was assayed (P<0. 01) but not when the reverse transcriptions were normalized to RNA content (P>0.05). The expression of beta-2 microglobulin increased up to two-fold when assayed from cDNA synthesized from non-normalized mRNA, but was unaffected by serum when the reverse transcriptions were normalized to mRNA. beta-2 Microglobulin expression was found to be directly proportional to the amount of mRNA that was present in non-normalized reverse transcription reactions. Thus, beta-2 microglobulin and 18S rRNA are suitable internal control genes in quantitative serum-stimulation studies, while beta-actin and GAPDH are not. The internal control gene needs to be properly validated when designing quantitative gene expression studies.

Quantitative reverse transcription-polymerase chain reaction to study mRNA decay: comparison of endpoint and real-time methods.

Four quantitative reverse transcription-PCR (RT-PCR) methods were compared to evaluate the time course of mRNA formation and decay. Mouse fibroblasts (NIH 3T3) transfected with the human beta-globin open reading frame/c-myc 3'-untranslated region chimeric gene under control of the c-fos promoter (fos-glo-myc) were used for serum-inducible transcription. The amount of fos-glo-myc mRNA, relative to beta-actin, was measured by quantitative, RT-PCR at various times following the addition of serum to serum-starved fibroblasts transfected with the chimeric gene. Both endpoint (band densitometry and probe hybridization) and real-time (SYBR green and TaqMan) PCR methods were used to assay the identical cDNA. The real-time methods produced a 4- to 5-log dynamic range of amplification, while the dynamic range of the endpoint assays was 1-log. The real-time and probe hybridization assays produced a comparable level of sensitivity that was considerably greater than band densitometry. The coefficient of variation from 22 replicate PCR reactions was 14.2 and 24.0% for the SYBR green and TaqMan detection, respectively, and 44.9 and 45.1% for the band densitometry and probe hybridization assays, respectively. The rank order for the values of r(2) obtained from the linear regression of the first-order mRNA decay plots was SYBR green > TaqMan > probe hybridization > band densitometry. Real-time PCR is more precise and displays a greater dynamic range than endpoint PCR. Among the real-time methods, SYBR green and TaqMan assays produced comparable dynamic range and sensitivity while SYBR green detection was more precise and produced a more linear decay plot than TaqMan detection.

Characterization of nitric oxide production following isolation of rat hepatocytes.

Freshly isolated suspensions of rat parenchymal liver cells (hepatocytes) produce large amounts of nitrite following isolation. Nitrite production was inhibited by the inducible nitric oxide synthase (iNOS) inhibitor aminoguanidine, as well as the transcription inhibitor actinomycin D. Increases in iNOS mRNA, protein, and activity levels correlated with the formation of nitrite. iNOS mRNA was first detectable 2 h after the onset of hepatocyte incubations and peaked at 4 h. These results indicate that nitrite formation is a result of the large scale production of nitric oxide (NO) by hepatocytes in response to the time-dependent induction of iNOS. NO production by hepatocytes was attenuated by pretreatment of rats with the Kupffer cell inhibitor, gadolinium chloride. Also, the addition of the endotoxin neutralizing agent, polymyxin B; the protein kinase inhibitor, staurosporine, and antioxidants to perfusion buffers and hepatocyte suspensions also decreased nitrite formation. Collectively, our results suggest that iNOS is induced in hepatocytes in response to the stresses generated during collagenase isolation procedures. The response appears to be triggered by a complex interaction between several different factors including Kupffer cell activation, reactive oxygen species generation, and endotoxin contamination of collagenase preparations.

Glutathione-dependent regulation of nitric oxide production in isolated rat hepatocyte suspensions.

Freshly isolated suspensions of rat parenchymal liver cells (hepatocytes) spontaneously produce large amounts of nitrite following collagenase isolation. Our previous studies indicate that nitrite production is associated with the expression of inducible nitric oxide synthase (iNOS) and reflects NO production. Depletion of glutathione (GSH) with diethylmaleate (DEM) inhibited nitrite production, and this inhibition was time-dependent. DEM was more effective in blocking nitrite production if it was added within the first 1 hr of the start of the incubation. The reducing agent dithiothreitol (DTT) and the alkylating agent ethyl methanesulfonate (EMS) also inhibited hepatocyte nitrite production, and this inhibition was also greatest if they were added within 1 hr of initiating the incubation. However, EMS added at 3 hr still reduced 6-hr nitrite production by about 70%. This reduction in nitrite production by EMS added at 3 hr may be due to the direct modification of thiol groups on the iNOS protein because we have determined that iNOS activity is inhibited by the sulfhydryl modifying reagent N-ethylmaleimide (NEM). Western blots also indicate that the iNOS protein is expressed when EMS is added at 3 hr. The addition of DEM, DTT, or EMS at 0 time greatly reduced the levels of cellular iNOS mRNA relative to controls as determined by quantitative RT-PCR. Based on our results with mRNA levels, both DTT and depletion of cellular GSH appear to inhibit the early signaling events leading to iNOS expression and suggest that the control of iNOS induction in hepatocytes is sensitive to the thiol redox status of the cell.