# Presentation: 128085

## Global Transcriptional Analysis of Desulfovibrio vulgaris in Response to Nitrite Stress

**Global Transcriptional Analysis of *****Desulfovibrio vulgaris***** in Response to Nitrite Stress**

**Qiang He****1****, Zhili He****1****, Liyou Wu****1****, Judy D. Wall****2****, Terry C. Hazen****3****, Matthew W. Fields****4****,** and **Jizhong Zhou****1**** **

**1****Oak Ridge National Laboratory, Oak Ridge, TN**
**2****University of Missouri, Columbia, MO**
**3****Lawrence Berkeley National Laboratory, Berkeley, CA **
**4****Miami University, Oxford, OH**

***Desulfovibrio vulgaris***** has been studied extensively for its potential in the bioremediation of heavy metals and radionuclides. Nitrate, a frequent environmental co-contaminant, has been reported to inhibit microbial sulfate and uranium reduction thereby posing a limitation on potential remediation activity. Growth experiments indicated that log-phase *****D. vulgaris***** sustained growth at nitrite concentrations below 2 mM with concurrent removal of nitrite. At nitrite concentrations above 5 mM, no significant growth and nitrite removal were observed. In contrast, growth occurred rapidly at nitrate concentrations higher than 10 mM, suggesting nitrite, an intermediate of nitrate reduction, is responsible for the inhibition. To assess the mechanism of nitrite resistance, genome-wide transcriptional profiling analysis was carried out following nitrite (2.5 mM) treatment. Whole-genome microarrays of *****D. vulgaris***** were constructed with 70mer oligonucleotides representing all 3584 ORFs in the 3.6 Mb genome. Transcripts highly up-regulated throughout the 3.5 h following nitrite shock included genes encoding a nitrite reductase, a hybrid cluster (Fe/S) protein, and a group of Fe****2+**** transport proteins. This evidence confirmed that the hybrid cluster protein’s potential involvement in nitrogen metabolism and oxidative stress. The induction of nitrite reductase genes were correlated to the simultaneous repression of nitrate reductase genes along with the induction of a nitrogen assimilation regulator, suggesting nitrite stress response is likely regulated via the nitrogen assimilation mechanism. In addition, iron limitation was evident upon nitrite treatment as indicated by the up-regulation of Fe****2+**** transporter genes and an iron-repressible flavordoxin gene. These results suggested that *****D. vulgaris***** has a well coordinated regulatory response to elevated nitrite levels, combining regulation on both oxidative stress and nitrogen metabolism. Comparison of transcriptional responses between different oxidative strtessors would provide further insight into the mechanisms of microbial survival and adaptation in environments relevant to bioremediation of contaminated DOE sites. **

***Genes highly Up-regulated under Nitrite Stress***

***Abstract***

***Acknowledgements***

**This research was funded by the U.S. Department of Energy (Office of Biological and Environmental Research, Office of Science) grants from the Genomes To Life Program. Oak Ridge National Laboratory is managed by University of Tennessee-Battelle LLC for the Department of Energy under contract DOE-AC05-00OR22725.**

_***Genes of N metabolism***_

***Summary***

***Microarray Construction***

***Genes highly Down-regulated under Nitrite Stress***

***Results***

***Results***

_***Iron Limitation***_

- Cover all ORFs in the genome with 3574 oligos, including 3471 (97.1%) unique probes and 103 (2.9%) probes which may cross-hybridize with other ORFs
- Genome size = 3.6 Mb.
- Oligo length = 70mer
- 10 ORFs have multiple copies. For those ORFs, only one probe for all replicates
- Negative controls: 10 oligo probes for 10 human genes and 10 oligos for 10 *Arabidopsis* genes without mRNA spiked
- Positive controls: different amounts of genomic DNA and probes for human genes and *Arabidopsis* genes with mRNA spiked in samples
- Each spot repeated once

_***Transcriptional Dynamic***_

**Most dramatic changes in the transcriptome were observed within 1.5 hours following nitrite addition**
**The number of genes differentially expressed decreased rapidly after 1.5 hours in accordance with the decrease in nitrite concentration**
**Maximum around 300 genes were up- and down-regulated following nitrite stress**

**Expected increase in nitrite reduction gene expression**
**Indications of coordination by N assimilation regulator**
**Nitrite reduction genes—Up**
**Nitrate reduction genes—Down**
**Nitrogenous substrate transport—Down**
**N assimilation regulator—Up**
_***Hybrid Cluster Protein***_
**Hybrid cluster protein was first discovered in *****D. vulgaris***
**Extremely active redox protein conserved in several other bacteria; no confirmed function**
**Suggested function:**
**Nitrogen metabolism**
**Oxygen stress**
**Highly induced along with another Fe-S cluster protein under nitrite stress**
**Comparison with oxygen stress will ascertain its function**

_***Oxidative stress Responsive Genes***_

**More than 20 putative oxidative stress related genes were moderately (2~3 fold) up-regulated**
**Terminal oxidases**
**The low affinity *****aa3*****-type terminal cytochrome *****c***** oxidase (*****cox*****) operon **
**The cytoplasmic non-energy conserving *****sor-rub-roo***** operon**
**Peroxidases**
***tpX*****—thiol peroxidase**
**DVU2247—antioxidant, ApC/Tsa family**
**Thiol reductases**
**Glutaredoxin**
***msrA***
**Iron homeostasis**
**Up-regulation of *****fur***
**Up-regulation of ferritin operon (Fe****3+**** uptake)**

**Indications of iron limitation under nitrite stress**
**Induction of Fe****2+**** transporter (*****feoB*****) operon**
**Induction of ferritin (*****ftn*****) operon for Fe****3+**** uptake**
**Up-regulation of flavordoxin gene, which is repressed by iron**

**Biotic nitrite reduction is the predominant mechanism of nitrite removal, compared to abiotic reduction by sulfide.**
**Growth of log-phase *****D. vulgaris***** cells was sustained with the addition of nitrite up to 2 mM, while growth ceased with nitrite higher than 5 mM.**
**The inability to growth with the presence of 5 mM nitrite coincided with the inability to removal nitrite.**
**Growth at nitrite less than 2 mM was accompanied by rapid reduction of nitrite.**

**Mechanism of nitrite detoxification by *****D. vulgaris***** is enzymatic nitrite reduction to ammonium.**
**Nitrite reduction is concentration-dependent: nitrite below 2 mM was rapidly reduced while concentration higher than 5 mM could not be efficiently removed by log-phase *****D. vulgaris***** cells.**
**The most dramatic changes in transcription occurred within 1.5 hours following nitrite treatment and the genes affected decreased rapidly beyond 1.5 h, in accordance with the decrease in nitrite concentration.**
**Genes involved in nitrogen metabolism were differentially expressed with the up-regulation of genes encoding the nitrite reductase and a hybrid cluster protein, and the down-regulation of nitrate reduction genes.**
**Nitrite stress also moderately induced a group of oxidative stress related genes.**
**Iron limitation in nitrite stressed cells was evident with the expression of iron transporter genes.**
**Nitrite stress also resulted in transient repression of ribosomal RNA genes and amino acid-tRNA synthetases.**

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