( ...)Amplification of the amoA Gene of Nitrosococcus oceani Bacteria Using ,,, Eppendorf MasterTaq

Kathryn L. Dudeck and Wendy A. Dustman
University of Georgia,
Department of Marine Sciences

Introduction

This application describes the use of the MasterTaq polymerase kit for improving PCR efficiency when studying functional gene presence. Using this polymerase, it was possible to amplify a 675 bp region of the amoA gene in lab-grown Nitrosococcus oceani, which has previously been met with many obstacles when utilizing other commercially available enzyme kits.

The Polymerase Chain Reaction (PCR) is a now-commonplace method used for making several copies of a specific gene for further manipulation, or for simply observing whether or not a specific gene is present.

Lab cultures of Nitrosococcus oceani and other ammonia-oxidizing bacteria (AOB) are notoriously difficult to study. They exhibit slow growth and therefore are easily contaminated by hetero t rophs. In addition to this hurdle, the gene used in this study is often found with three clustered subunits (amoA, amoB, and amoC) within some members of AOB (Alzerecca et. al, Sayavedra-Soto et. al). The gene lengths are remarkably similar; there f o re, the study of a single gene in
this area of the chromosome can be quite challenging.

Materials and Methods

Bacterial Preparation
Nitrosococcus oceani (ATCC 19707) was cultured in ATCC Medium 928 in a shaking incubator (100 rpm) at 27 C. Using 12.5% K2CO3, the pH level was maintained at ~8.3. Twenty-five milliliters were extracted and purified using a Qiagen blood Mini Prep Kit.

PCR Amplification
Two bacterial primers, A189, GGN GAC TGG GAC TCC TGG (Holmes et. al) and AmoA2-R, CCC CTC KGS AAA GCC TTC TTC (Rotthauwe et. al), were utilized to amplify a 675 bp region of the amoA gene of the cultures. PCR was performed as follows: 2 l of each primer (10 M), 1 l of N. oceani culture, 2.5 l of 10X buffer, 2 l of 2.5 mM dNTP, 2.5 U of Taq polymerase, and 0-5 l of 5X TaqMaster. This mixture was adjusted to a final volume of 25 l using
sterile water. A negative control (no DNA) was also amplified. Amplification of the target gene was carried out via the following program: 94 C for 2 minutes, 35 cycles of 94 C for 1 minute, 56 C for 1 minute, and 72 C for 1 minute, and a final extension step of 56 C for 5 minutes.

Visualization
PCR products were run in a 2.0% agarose gel and stained with ethidium bromide for visualization.

Results and Discussion

The 675 bp region of the amoA gene was successfully amplified using the Eppendorf MasterTaq polymerase kit. Varying the amount of TaqMaster thermostabilizer included in the PCR reaction had a minimal effect on the band obtained from N. oceani. The addition of 2.5 l of TaqMaster gave the most favorable results (Lane 4), though bands were visible at all concentrations of TaqMaster (Lanes 3 and 5). The additional bands seen in the N. oceani lanes are most likely the amo subunits. Internal probing after PCR verifies that the band seen at approximately 675 bp is indeed amoA (data not shown). Eppendorfs MasterTaq polymerase kit is a beneficial enzyme system to use when attempting to study known laboratory cultures of ammonia-oxidizing bacteria.

Figure 1: The use of MasterTaq for amplification of the amoA gene in two ammonia-oxidizing bacterial cultures
Lane 1 - Molecular weight marker VIII
Lane 2 - Negative control (no DNA)
Lane 3 - Nitrosococcus oceani with no Ta q M a s t e r
Lane 4 - Nitrosococcus oceani with 2.5 l Ta q M a s t e r
Lane 5 - Nitrosococcus oceani with 5 l Ta q M a s t e r

References

Alzerecca, J.J., J.M. Norton, and M.G. Klotz. 1999. The amo operon in marine, ammonia-oxidizing gamma-proteobacteria. FEMS Microbiology Letters 180:21-29.

Holmes, A. J., A. Costello, M. E. Lidstrom, and J. C. Murrell. 1995. Evidence that particulate methane monooxygenase and ammonia monooxygenase may be evolutionarily related. FEMS Microbiology Letters. 132:203-208.

Rotthauwe, J. H., K. P. Witzel, and W. Liesack. 1997. The ammonia monooxygenase structural gene amoA as a functional marker: molecular fine-scale analysis of natural ammonia-oxidizing populations. Applied and Environmental Microbiology. 63:4704-4712.

Sayavedra-Soto, L.A., N.G. Hommes, J.J. Alzerreca, D.J. Arp, J.M. Norton and M.G. Klotz. 1998. Transcription of the amoC, amoA and amoB genes in Nitrosomonas europaea and Nitrosospira sp. NpAV. FEMS Microbiology Letters 167:81-88.

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