Distinctive microbial community and genome structure in coastal seawater from a human-made port and nearby offshore island in northern Taiwan facing the Northwestern Pacific Ocean.

Pollution in human-made fishing ports caused by petroleum from boats, dead fish, toxic chemicals, and effluent poses a challenge to the organisms in seawater. To decipher the impact of pollution on the microbiome, we collected surface water from a fishing port and a nearby offshore island in northern Taiwan facing the Northwestern Pacific Ocean. By employing 16S rRNA gene amplicon sequencing and whole-genome shotgun sequencing, we discovered that Rhodobacteraceae, Vibrionaceae, and Oceanospirillaceae emerged as the dominant species in the fishing port, where we found many genes harboring the functions of antibiotic resistance (ansamycin, nitroimidazole, and aminocoumarin), metal tolerance (copper, chromium, iron and multimetal), virulence factors (chemotaxis, flagella, T3SS1), carbohydrate metabolism (biofilm formation and remodeling of bacterial cell walls), nitrogen metabolism (denitrification, N2 fixation, and ammonium assimilation), and ABC transporters (phosphate, lipopolysaccharide, and branched-chain amino acids). The dominant bacteria at the nearby offshore island (Alteromonadaceae, Cryomorphaceae, Flavobacteriaceae, Litoricolaceae, and Rhodobacteraceae) were partly similar to those in the South China Sea and the East China Sea. Furthermore, we inferred that the microbial community network of the cooccurrence of dominant bacteria on the offshore island was connected to dominant bacteria in the fishing port by mutual exclusion. By examining the assembled microbial genomes collected from the coastal seawater of the fishing port, we revealed four genomic islands containing large gene-containing sequences, including phage integrase, DNA invertase, restriction enzyme, DNA gyrase inhibitor, and antitoxin HigA-1. In this study, we provided clues for the possibility of genomic islands as the units of horizontal transfer and as the tools of microbes for facilitating adaptation in a human-made port environment.

Identification and Expression Analyses of the Nitrate Transporter Gene (NRT2) Family Among Skeletonema species (Bacillariophyceae).

High-affinity nitrate transporters are considered to be the major transporter system for nitrate uptake in diatoms. In the diatom genus Skeletonema, three forms of genes encoding high-affinity nitrate transporters (NRT2) were newly identified from transcriptomes generated as part of the marine microbial eukaryote transcriptome sequencing project. To examine the expression of each form of NRT2 under different nitrogen environments, laboratory experiments were conducted under nitrate-sufficient, ammonium-sufficient, and nitrate-limited conditions using three ecologically important Skeletonema species: S. dohrnii, S. menzelii, and S. marinoi. Primers were developed for each NRT2 form and species and Q-RT-PCR was performed. For each NRT2 form, the three Skeletonema species had similar transcriptional patterns. The transcript levels of NRT2:1 were significantly elevated under nitrogen-limited conditions, but strongly repressed in the presence of ammonium. The transcript levels of NRT2:2 were also repressed by ammonium, but increased 5- to 10-fold under nitrate-sufficient and nitrogen-limited conditions. Finally, the transcript levels of NRT2:3 did not vary significantly under various nitrogen conditions, and behaved more like a constitutively expressed gene. Based on the observed transcript variation among NRT2 forms, we propose a revised model describing nitrate uptake kinetics regulated by multiple forms of nitrate transporter genes in response to various nitrogen conditions in Skeletonema. The differential NRT2 transcriptional responses among species suggest that species-specific adaptive strategies exist within this genus to cope with environmental changes.

Evaluation of the Relationship Between the 18S rRNA/rDNA Ratio and Population Growth in the Marine Diatom Skeletonema tropicum via the Application of an Exogenous Nucleic Acid Standard.

Ribosomal RNA (rRNA) has been regarded as a proxy for metabolic activity and population growth in microbes, but the limitations and assumptions of this approach should be better defined, particularly in eukaryotic microalgae. In this study, the 18S rRNA/rDNA ratio of a marine diatom, Skeletonema tropicum, was examined in batch and semi-continuous cultures subjected to low nitrogen and phosphorus treatments at a temperature of 20 °C. In the semi-continuous cultures, the measured 18S rRNA/rDNA ratio ranged from 4.0 × 102 to 5.0 × 103 , and the logarithmic form of this ratio increased linearly with the population growth rate under both low nitrogen and low phosphorus conditions. In batch cultures grown under low nitrogen or low phosphorus conditions, log (rRNA/rDNA) also increased linearly with growth rate when the latter ranged between -0.4 and 1.5 day-1 . The 18S rRNA/rDNA ratios of Skeletonema sampled from in the southern East China Sea were substantially lower than measured from laboratory cultures. Among the field samples, ratios obtained at a coastal station were higher than those obtained farther offshore. These results imply higher growth rate at the coastal station, but the influences of other factors, such as cell size and temperature, cannot be ruled out.

The expression of nitrate transporter genes reveals different nitrogen statuses of dominant diatom groups in the southern East China Sea.

In this study, the mRNA levels of the Nrt2 nitrate transporter gene were used as a molecular indicator of nitrogen status in two dominant diatom groups, Skeletonema and Chaetoceros, which inhabit the southern East China Sea (ECS). To accurately interpret the abundance of Nrt2 transcripts in situ, maximum and minimum expression levels were determined under conditions of nitrogen deprivation and ammonium addition, respectively. In August 2010, Nrt2 transcript levels in Skeletonema at the inner shelf region exhibited a mean of 111 mmole/(mole EFL); at the mid-shelf region, the mean Nrt2 mRNA levels were 298 mmole/(mole EFL), which was very close to the maximum levels observed under nitrogen starvation. By contrast, the Nrt2 transcript levels in Chaetoceros were low at all of the shelf locations, except at one station in the mid-shelf region. The cross-shelf mean was 2.86 mmole/(mole EFL), which was similar to the expression levels observed in cultured Chaetoceros under conditions of sufficient ammonium. Similar expression patterns were observed in diatoms in the southern ECS in June 2011, but the Nrt2 transcript levels in Skeletonema at the inner shelf region were reduced to a mean of 28.6 mmole/(mole EFL). Regression analysis indicated that cell abundance and Nrt2 expression were closely related to the nutricline depth in the coastward half of the southern ECS for Skeletonema but not for Chaetoceros. These results indicate that the evaluated species differ in nitrogen status, which may reflect their evolutionary strategies to survive in a fluctuating marine environment.

Quantification of diatom gene expression in the sea by selecting uniformly transcribed mRNA as the basis for normalization.

To quantify gene expressions by quantitative reverse transcription-PCR (Q-RT-PCR) in natural diatom assemblages, it is necessary to seek a biomass reference specific to the target species. Two housekeeping genes, TBP (encoding the TATA box-binding protein) and EFL (encoding the translation elongation factor-like protein), were evaluated as candidates for reference genes in Q-RT-PCR assays. Transcript levels of TBP and EFL were relatively stable under various test conditions including growth stages, light-dark cycle phases, and nutrient stresses in Skeletonema costatum and Chaetoceros affinis, and TBP expression was more stable than that of EFL. Next, the sequence diversity of diatom assemblages was evaluated by obtaining 32 EFL and 29 TBP homologous gene fragments from the East China Sea (ECS). Based on sequence alignments, EFL and TBP primer sets were designed for Chaetoceros and Skeletonema groups in the ECS. An evaluation of primer specificity and PCR efficiency indicated that the EFL primer sets performed better. To demonstrate the applicability of EFL primer sets in the ECS, they were employed to measure mRNA levels of the FcpB (fucoxanthin-chlorophyll protein) gene in diatoms. The results correctly revealed prominent diel variations in FcpB expression and confirmed EFL as a good reference gene.

Diversity of phytoplankton nitrate transporter sequences from isolated single cells and mixed samples from the East China Sea and mRNA quantification.

The transcript abundances of nitrate transporter genes (Nrt2) were proposed as potential markers for nitrogen deficiency in marine diatoms. To correctly quantify diatom Nrt2 mRNA in the East China Sea (ECS), we utilized both mixed-species sequencing and single-cell PCR to expand the sequence database for this region. Using the single-cell method of PCR, 9 new diatom Nrt2 sequences belonging to 5 genera, the Nrt2 sequences of which have never been reported before, were obtained. On the other hand, 291 sequences homologous to Nrt2 were retrieved from mixed-species sequencing using degenerate primers, and these sequences were clustered into 12 major groups according to a phylogenetic analysis. Based on sequence alignments, 11 pairs of group-specific PCR primers were designed to detect Nrt2 mRNA levels in the ECS, and 3 of these primer pairs showed high specificity to target species. In ECS phytoplankton samples, environmental RNA was amplified via antisense RNA amplification followed by cDNA production. Subsequently, Nrt2 transcript levels were readily detected using quantitative PCR. Our results indicated that investigating sequence diversity followed by careful primer design and evaluation is a good strategy to quantify the expression of genes of ecologically important phytoplankton.

ESTABLISHMENT OF MINIMAL AND MAXIMAL TRANSCRIPT LEVELS FOR NITRATE TRANSPORTER GENES FOR DETECTING NITROGEN DEFICIENCY IN THE MARINE PHYTOPLANKTON ISOCHRYSIS GALBANA (PRYMNESIOPHYCEAE) AND THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE)(1).

Nitrate transporter genes (Nrt2) encode high-affinity nitrate transporters in marine phytoplankton, and their transcript levels are potential markers of nitrogen deficiency in eukaryotic phytoplankton. For the proper interpretation of measured Nrt2 transcript abundances, a relative expression assay was proposed and tested in Isochrysis galbana Parke (Prymnesiophyceae) and Thalassiosira pseudonana (Hust.) Hasle et Heimdal (Bacillariophyceae). The minimal transcript levels of Nrt2 genes were achieved by the addition of 100 µM ammonium, which led to a rapid decline in Nrt2 transcripts in 10-30 min. Experiments using a concentration series revealed that the effective dosage of ammonium to create a minimal transcript level of ∼1 µmol · mol(-1) 18S rRNA was ≥25 µM in both species. On the other hand, the addition of l-methionine sulfoximine (MSX), an inhibitor of glutamine synthetase, enhanced the Nrt2 transcript level in I. galbana but did not affect that in T. pseudonana. Nitrogen deprivation was used as an alternative means to create maximal Nrt2 transcript levels. By transferring cells into N-free medium for 24 h, Nrt2 transcript levels increased to ∼90 µmol · mol(-1) 18S rRNA in I. galbana, and to ∼800 µmol · mol(-1) 18S rRNA in T. pseudonana. The degree of nitrogen deficiency thus can be determined by comparing original Nrt2 transcript levels with the minimal and maximal levels.