# Presentation: 826718

## Transcriptional Analysis and Characterization of Desulfovibrio vulgaris Cells from Logarithmic and Stationary Phase Growth

- M.E. Clark1, Z. He2, Q. He2, L. Wu2, J.D. Wall3, J. Zhou2 and M. W. Fields1
- 3Miami University, Oxford, OH, 2Oak Ridge National Laboratory, Oak Ridge, TN; 3University of Missouri, Columbia, MO;

- 							        **Abstract**
*Desulfovibrio vulgaris* is* *a -*Proteobacterium* with a genome size of 3.6 Mb and 3584 predicted ORFs. The whole-genome microarrays of *D. vulgaris* have been constructed using 70mer oligonucleotides. All ORFs in the genome were represented with 3471 (97.1%) unique probes and 103 (2.9%) non-specific probes.  In preparation for use of the experimental microarrays, artificial probes and targets were designed to assess specificity and sensitivity and identify optimal hybridization conditions for oligonucleotide microarrays.  The results indicated that for 50mer and 70mer oligonucleotide arrays, hybridization at 45oC to 50oC, washing at 37oC and a wash time of 2.5 to 5 minutes obtained specific and strong hybridization signals.  In order to evaluate the performance of the experimental microarrays, growth conditions were selected that were expected to give significant hybridization differences for different sets of genes.  The initial evaluations were performed using *D. vulgaris* cells grown at logarithmic and stationary phases.  Transcriptional analysis of *D. vulgaris* cells sampled during logarithmic phase growth indicated that 25% of annotated ORFs were up-regulated and 3% of annotated ORFs were down-regulated compared to stationary phase cells. The up-regulated genes included ORFs predicted to be involved with acyl chain biosynthesis, amino acid ABC transporter, translational initiation factors, and ribosomal proteins.  In addition, biologically relevant ORFs (i.e., formate dh, lactate dh, phosphotransacetylase, sulfite reductase, lactate permease) had increased expression in logarithmic phase cells.  However, several of the highly up-regulated ORFs were annotated as conserved hypothetical proteins.  Protein and carbohydrate levels were monitored over time for cells grown in LS, and both the carbohydrate and carbohydrate to protein ratio increased when the cultures were in late-logarithmic phase growth.  Genome annotation predicts that *D*. *vulgaris* has all needed proteins for the biosynthesis and utilization of glycogen.  In addition, *D*. *vulgaris* is predicted to have a fructose-2,6-bisphosphatase/kinase, which is much more common in eukaryotes and is used to control carbon flow via allosteric regulation of the phosphofructokinase and bisphosphatase.  In LS-grown cells, the phosphoglucomutase and UDP-glucose pyrophosphorylase were up-expressed 9- to 29-fold in logarithmic cells, the glycogen phosphorylase was up-expressed 7-fold, and the glucanotransferase was up-expressed 2-fold.  A putative glucokinase was up-expressed 1.5-fold and the glycogen branching enzyme was up-expressed 2.7-fold in stationary phase cells.  The transient increase in the carbohydrate to protein ratio was also observed in cells grown LS4D medium.  Methods have been developed to differentiate internal and external carbohydrate levels, and results suggest that *D*. *vulgaris *cells deplete internal carbohydrate levels during the early growth cycle and then increase the internal levels during late-exponential phase growth.  Little is known about glycogen levels with respect to the growth phase in *D*. *vulgaris*, or the possible role of these levels during stress and survival.  Future work includes the whole-genome expression comparison of logarithmic- and stationary-cells grown in LS4D medium in relation to protein, carbohydrate, sulfate, and lactate levels, and the possible role of glycogen during heavy metal stress.

**Conclusions**:
-   The microarrays worked well when whole-genome expression patterns were compared for logarithmic and stationary phase cells grown in LS
-   Future work includes RT-PCR to confirm expression levels of selected ORFs
-   The initial results suggested that *D*. *vulgaris* might produce polyamines and possible pathways are being investigated
-   Genomic analyses indicate that *D*. *vulgaris* does not have the mevalonate pathway for isoprenoid biosynthesis, but does have the predicted proteins for the deoxy-xylulose pathway.  The microarray data indicated that different ORFs for this pathway were differentially expressed between logarithmic and stationary phase cells
-   Several predicted molecular chaperone proteins were up-regulated in stationary phase growth
-   An ORF with a predicted flocculin domain was up-regulated in stationary phase cells and this domain is uncommon in prokaryotes
-   A hypothetical protein that was up-regulated in stationary phase cells, which is conserved with *Desulfovibrio* *desulfuricans* G20, has low homology with a proteophosphoglycan from *Leischmania*
-   As cells entered stationary phase growth, a transient increase in total carbohydrate levels was observed for cells grown in both LS and LS4D
-   Methods were compared for the extraction of external carbohydrate for *D*. *vulgaris* cultures
-   *D*. *vulgaris* cultures increased glycogen levels as the cells entered stationary phase growth
-   Future work includes the whole-genome expression analysis over time of batch cultures
-   Determine possible roles for glycogen in survival and stress response in *D*. *vulgaris*

**Up-Expressed ORFs During Logarithmic Phase**

| TIGR ORF Number | ORF Annotation | Relative Change in Expression |
| --- | --- | --- |
| DVU2481 | Formate dehydrogenase () | 9.4 |
| DVU2483 | Cytochrome C (III) | 8.0 |
| DVU2484 | Cytochrome bd | 22 |
| DVU2485 | Membrane protein | 3.3 |
| DVU2783 | Membrane protein | 19 |
| DVU2784 | Lactate dehydrogenase | 19 |
| DVU3025 | Pyruvate-ferredoxin reductase | 17 |
| DVU3026 | Lactate permease | 3.7 |
| DVU3027 | FAD/FMN dehydrogenase | 16 |
| DVU3028 | Fe-S oxidoreductase | 5.2 |
| DVU3029 | Phosphotransacetylase | 23 |
| DVU3030 | Acetate kinase | 31 |
| DVU3031 | Phosphotransacetylase | 7.4 |
| DVU3033 | Ferredoxin-like protein | 11 |
| DVU0402 | dsrA | 11 |
| DVU0403 | dsrB | 20 |
| DVU0404 | dsrD | 31 |
| DVU2451 | Lactate permease | 13 |

| TIGR ORF Number | ORF Annotation | Relative Change in Expression |
| --- | --- | --- |
| DVU2988 | Phage shock protein
(suppresses σ54 transcription) | 25 |
| DVU0419 | Carboxynorspermidine dc | 70 |
|  |  |  |
| DVU0531 | Fe-S oxidoreductase (hmcF) | 2.2 |
| DVU0533 | hmcD | 11 |
| DVU0536 | Cytochrome c (hmcA) | 2 |
| DVU1454 | Cytidyltransferase; cyclodiphosphate synthase | 66 |
| DVU1976 | Chaperone (60 kDa) | 2.8 |
| DVU1977 | Chaperone (10 kDa, groES) | 7.3 |
| DVU2441 | Heat shock protein | 25 |
| DVU2442 | Heat shock protein | 3.4 |
| DVU2466 | Flocculin domain protein | 8.6 |
| DVU1875 | DafA  | 2.5 |
| DVU1876 | DnaJ (chaperone) | 3.9 |

**Up-Expressed ORFs During Stationary Phase**

**Logarithmic and Stationary Phase Cells Grown in LS medium at 37****°****C**

|  | Up-Expressed ORFs in Log Phase 
(cells grown in LS) |  |
| --- | --- | --- |
| TIGR ORF Number | ORF Annotation | Relative Change in Expression |
| DVU1428 | Phosphoglucomutase | 29 |
| DVU1283 | UDP-Glc pyrophosphorylase/undylyltransferase | 29 |
| DVU2322 |  | - |
| DVU2244 | Glycogen synthase | - |
| DVU2349 | Glycogen phosphorylase | 7 |
| DVU3148 | Glucano transferase | 1.8 |
| DVU2239 | Glucosidase | - |
| DVU2061 | Phosphofructokinase | - |
| DVU1539 | 1;6 Bisphosphatase class II | - |
| DVU1841 |  | - |
| DVU1550 | 2;6 Bisphosphotase | - |
| DVU3147 | 2;6 Bisphosphokinase | - |
| DVU1907 | UDP-glucose 6-dehydrogenase | 4.2 |
|  | Up-Expressed ORFs in Stationary Phase |  |
| DVU1035 | Glucokinase | 1.5 |
| DVU2241 | Isoamylase N-terminal domain protein | - |
| DVU0938 | (1,4-alpha glucan branching enzyme) | 2.7 |

**Method comparison for the determination of external carbohydrate**.  First, NaOH- and EDTA-based extraction methods were tested, but as previously reported, significant cell lysis occurred.  A previous study had used sodium chloride and formaldehyde, but these reagents caused interference in three different carbohydrate assays (Phenol, Anthrone, and Cysteine).    Extraction of external carbohydrate via ultracentrifugation and Zwittergent 3-14 revealed the removal of approximately 55% of total carbohydrate present in control cells, *Klebsiella pneumoniae*.  Protein concentrations from the cell pellets before and after the Zwittergent wash did not differ significantly (98% recovery), and this result indicated that a minimal amount of carbohydrate was being released from the cells due to cell lysis.  Previous reports have reported minimal cell lysis with the ultracentrifugation method, but  the ultracentrifugation method requires a larger sample volume.

- Carbohydrate levels increased as the cells entered stationary phase growth, and as observed in previous cultures, peaked approximately 40 h after inoculation.  Interestingly, when the cells were grown in LS medium, the carbohydrate levels increased at a similar time in the growth cycle (late-exponential).  However, the maximum carbohydrate to protein ratio for LS-grown cells was 5-fold lower than for LS4D-grown cells.  Preliminary analysis of external verses internal carbohydrate levels using the Zwittergent 3-14 detergent wash indicated that internal carbohydrate levels increased in accordance with the observed spike in total carbohydrates. This result indicated that an increase in glycogen could account for a majority of the increase in carbohydrate levels.  The results also indicated that the increase in glycogen was transient, which could be a response in preparation for stationary phase.

**Growth in LS4D at 30****°****C**.  Protein levels increased and sulfate levels decreased over time, and stationary phase growth initiated approximately when the sulfate levels remained constant.  As the cells entered stationary phase, carbohydrate levels increased, and peaked at approximately 250 ug/ml glucose equivalents (anthrone).  After 10 h, the carbohydrate levels decreased to a similar level that was observed during exponential phase growth.  The carbohydrate to protein ratio displayed a similar response during batch growth.  The results suggested that the cells increase carbohydrate content as a possible response to stationary phase growth.  Previous work in *Desulfovibrio* spp. has indicated that different species maintain varying levels of glycogen and exo-polysaccharide (EPS) levels.  However, little is known about glycogen and EPS in *D*. *vulgaris* Hildenbourough, and the possible roles for glycogen and/or EPS in survival and stress response.  Methods were then tested to elucidate if the spike in carbohydrate was internal or external.