Depth distributions of alkaline phosphatase and phosphonate utilization genes in the North Pacific Subtropical Gyre

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1 The following supplement accompanies the article Depth distributions of alkaline phosphatase and phosphonate utilization genes in the North Pacific Subtropical Gyre Haiwei Luo 1, *, **, Hongmei Zhang 2, **, Richard A. Long 1,3, Ronald Benner 1,3 1 Department of Biological Sciences, University of South Carolina, Columbia, South Carolina 29208, USA 2 Department of Epidemiology and Biostatistics, University of South Carolina, Columbia, South Carolina 29208, USA 3 Marine Science Program, University of South Carolina, Columbia, South Carolina 29208, USA **These authors contributed equally to this article * hluo2006@gmail.com Aquatic Microbial Ecology 62:61 69 (2011) INTRODUCTION This supplement provides a rationale in support of constraints upon rejection of hypothesis 2; seed query information (Table S1); enzyme (C-P lyase, C-P hydrolase and alkaline phosphatase) peptide identification and depth distribution of their coding genes (Tables S2 to S7); and information on the effect of sample size on gene proportions in samples (Fig. S1). MATERIALS AND METHODS Database download. The bioinformatics databases were downloaded from the following websites in April The NCBI CDD was downloaded from NCBI: ftp://ftp.ncbi.nlm.nih.gov/pub/mmdb/cdd/; the NCBI non-redundant database was downloaded from NCBI: ftp://ftp.ncbi.nih.gov/blast/db/; one NPSG depth-variable metagenomic database (DeLong et al. 2006) was downloaded from CAMERA: The file name is node fasta. An explanation of the claim if hypothesis 2 cannot be rejected through direction 2, it cannot be rejected through direction 1 either. We use N 130 to denote the current sample size at depth 130 m or deeper. A larger sample size is denoted as: with It is sufficient to explain the equivalence between the 2 directions if we miscounted the executor genes by 1 in the current sample (i.e. we actually should have observed 1 such gene), since it may not be realistic to assume that many genes had been miscounted as stated in the text. A 95% confidence interval can be set up for the situation above. The lower bound is given as: If the above confidence interval includes zero, then the lower bound must be negative, i.e.: This gives:, i.e. Now, if the sample size increases to, since and we then have: In other words, the confidence interval set up based on the new sample size will include zero as well. Therefore, if hypothesis 2 cannot be rejected through direction 2, it cannot be rejected through direction 1 either.

2 Table S1. Seed query information. Protein name Seed query NCBI accession Source organism PhnG AAC YP_ PhnH AAC YP_ PhnI AAC YP_ PhnJ AAC YP_ PhnM AAC ACB PhnK AAT YP_ PhnL AAC YP_ PhnA a AAC ACA PhnX b Q9I433.1 ZP_ PhoA YP_ ABP PhoD NP_ YP_ PhoX ABL YP_ a phna: phosphonoacetate hydrolase b phnx: phosphonoacetaldehyde hydrolase (phosphonatase) Pseudomonas fluorescens Unclassified marine bacteria Pseudomonas aeruginosa Bacteroides fragilis Chlorobium phaeovibrioides Bacillus subtilis Shewanella loihica Pasteurella multocida Synechococcus sp.wh 7803 Table S2. C-P lyase (PhnM, PhnJ, PhnG, PhnH, PhnI, PhnK, PhnL) peptide ID identified from metagenomic database in DeLong et al. (2006). ID Depth (m) Gene JCVI_PEP_ phng JCVI_PEP_ phng JCVI_PEP_ phnh JCVI_PEP_ phnh JCVI_PEP_ phni JCVI_PEP_ phni JCVI_PEP_ phni JCVI_PEP_ phnk JCVI_PEP_ phnk JCVI_PEP_ phnk JCVI_PEP_ phnk JCVI_PEP_ phnk JCVI_PEP_ phnk JCVI_PEP_ phnk JCVI_PEP_ phnk JCVI_PEP_ phnk JCVI_PEP_ phnk JCVI_PEP_ phnk JCVI_PEP_ phnk JCVI_PEP_ phnk 2

3 JCVI_PEP_ phnk JCVI_PEP_ phnk JCVI_PEP_ phnk JCVI_PEP_ phnk JCVI_PEP_ phnk JCVI_PEP_ phnl JCVI_PEP_ phnl JCVI_PEP_ phnl JCVI_PEP_ phnl JCVI_PEP_ phnl JCVI_PEP_ phnl JCVI_PEP_ phnl JCVI_PEP_ phnl JCVI_PEP_ phnl JCVI_PEP_ phnl JCVI_PEP_ phnl JCVI_PEP_ phnl JCVI_PEP_ phnl JCVI_PEP_ phnl JCVI_PEP_ phnl JCVI_PEP_ phnl Table S3. C-P hydrolase (PhnA, PhnX) peptide ID identified from metagenomic database in DeLong et al. (2006). ID Depth (m) Gene JCVI_PEP_ phna JCVI_PEP_ phna JCVI_PEP_ phna JCVI_PEP_ phna JCVI_PEP_ phna JCVI_PEP_ phna JCVI_PEP_ phna JCVI_PEP_ phna JCVI_PEP_ phna JCVI_PEP_ phna JCVI_PEP_ phna JCVI_PEP_ phna JCVI_PEP_ phna JCVI_PEP_ phna JCVI_PEP_ phna JCVI_PEP_ phna JCVI_PEP_ phnx JCVI_PEP_ phnx JCVI_PEP_ phnx JCVI_PEP_ phnx 3

4 Table S4. Alkaline phosphatase (PhoA, PhoX, PhoD) peptide ID identified from metagenomic database in DeLong et al. (2006). ID Depth (m) Gene JCVI_PEP_ phod JCVI_PEP_ phod JCVI_PEP_ phod JCVI_PEP_ phod JCVI_PEP_ phod JCVI_PEP_ phod JCVI_PEP_ phod JCVI_PEP_ phod JCVI_PEP_ phod JCVI_PEP_ phod JCVI_PEP_ phod JCVI_PEP_ phod JCVI_PEP_ phod JCVI_PEP_ phod JCVI_PEP_ phod JCVI_PEP_ phod JCVI_PEP_ phod JCVI_PEP_ phod JCVI_PEP_ phod JCVI_PEP_ phod JCVI_PEP_ phod JCVI_PEP_ phod JCVI_PEP_ phod JCVI_PEP_ phod JCVI_PEP_ phod JCVI_PEP_ phod JCVI_PEP_ phod JCVI_PEP_ phod JCVI_PEP_ phod JCVI_PEP_ phod JCVI_PEP_ phod JCVI_PEP_ phod JCVI_PEP_ phod JCVI_PEP_ phox JCVI_PEP_ phox JCVI_PEP_ phox JCVI_PEP_ phox JCVI_PEP_ phox JCVI_PEP_ phox JCVI_PEP_ phox JCVI_PEP_ phox JCVI_PEP_ phox JCVI_PEP_ phox JCVI_PEP_ phox JCVI_PEP_ phox JCVI_PEP_ phox JCVI_PEP_ phox 4

5 JCVI_PEP_ phox JCVI_PEP_ phox JCVI_PEP_ phox JCVI_PEP_ phox JCVI_PEP_ phox JCVI_PEP_ phox JCVI_PEP_ phox JCVI_PEP_ phoa JCVI_PEP_ phoa JCVI_PEP_ phoa JCVI_PEP_ phoa Table S5. The depth distribution of C-P lyase genes expressed as a percentage of organisms that contain the gene of interest, provided that reca is a single-copy gene in bacterial genomes. Gene 10 m 70 m 130 m 200 m 500 m 770 m 4000 m phnm a phnj a phnh a phng a phni a phnk b phnl b reca a phng, phnh, phni, phnj & phnm are executor genes b phnk & phnl are transporter genes Table S6. The depth distribution of C-P hydrolase genes, expressed as a percentage of organisms that contain the gene of interest, provided that reca is a single-copy gene in bacterial genomes. Gene 10 m 70 m 130 m 200 m 500 m 770 m 4000 m phna a phnx b reca a phna: phosphonoacetate hydrolase gene b phnx: phosphonoacetaldehyde hydrolase (phosphonatase) gene Table S7. The depth distribution of alkaline phosphatase genes expressed as a percentage of organisms that contain the gene of interest, provided that reca is a single-copy gene in bacterial genomes. Gene 10 m 70 m 130 m 200 m 500 m 770 m 4000 m phoa phod phox reca

6 Fig. S1. The effect of sample size (total number of genes sampled) on sample proportions (p; the proportion of C-P lyase genes); this is a simulation study based on assumed binomial distribution of C-P lyase genes. The true proportion is assumed to be ( ), the observed sample proportion in the upper 70 m; it is represented by a solid horizontal line in the figure LITERATURE CITED DeLong EF, Preston CM, Mincer T, Rich V and others (2006) Community genomics among stratified microbial assemblages in the ocean's interior. Science 311:

SUPPLEMENTARY INFORMATION

Supplementary information S1 (box). Supplementary Methods description. Prokaryotic Genome Database Archaeal and bacterial genome sequences were downloaded from the NCBI FTP site (ftp://ftp.ncbi.nlm.nih.gov/genomes/all/)