ABSTRACT
As the most abundant d-amino acid (DAA) in the ocean, d-alanine (d-Ala) is a key component of peptidoglycan in the bacterial cell wall. However, the underlying mechanisms of bacterial metabolization of d-Ala through the microbial food web remain largely unknown. In this study, the metabolism of d-Ala by marine bacterium Pseudoalteromonas sp. strain CF6-2 was investigated. Based on genomic, transcriptional, and biochemical analyses combined with gene knockout, d-Ala aminotransferase was found to be indispensable for the catabolism of d-Ala in strain CF6-2. Investigation on other marine bacteria also showed that d-Ala aminotransferase gene is a reliable indicator for their ability to utilize d-Ala. Bioinformatic investigation revealed that d-Ala aminotransferase sequences are prevalent in genomes of marine bacteria and metagenomes, especially in seawater samples, and Gammaproteobacteria represents the predominant group containing d-Ala aminotransferase. Thus, Gammaproteobacteria is likely the dominant group to utilize d-Ala via d-Ala aminotransferase to drive the recycling and mineralization of d-Ala in the ocean.
IMPORTANCE As the most abundant d-amino acid in the ocean, d-Ala is a component of the marine DON (dissolved organic nitrogen) pool. However, the underlying mechanism of bacterial metabolization of d-Ala to drive the recycling and mineralization of d-Ala in the ocean is still largely unknown. The results in this study showed that d-Ala aminotransferase is specific and indispensable for d-Ala catabolism in marine bacteria and that marine bacteria containing d-Ala aminotransferase genes are predominantly Gammaproteobacteria widely distributed in global oceans. This study reveals marine d-Ala-utilizing bacteria and the mechanism of their metabolization of d-Ala. The results shed light on the mechanisms of recycling and mineralization of d-Ala driven by bacteria in the ocean, which are helpful in understanding oceanic microbial-mediated nitrogen cycle.
KEYWORDS: d-alanine, d-Ala, d-alanine catabolism, d-alanine aminotransferase, alanine racemase, marine bacteria
INTRODUCTION
Except for glycine, the canonical proteinogenic amino acids are chiral and exist in either d or l form, which are enantiomers. d-Amino acids (DAAs) have been detected in diverse environments such as soil, rivers, lakes, and oceans (1–4). DAAs are predominant in bacterial peptidoglycan and some other prokaryotic compounds, such as capsules, lipopolysaccharides, siderophores, and antimicrobial peptides (5–9). In the ocean, DAAs are important components of the dissolved organic matter (DOM) pool (10). As a matter of fact, d-alanine (d-Ala), d-glutamate (d-Glu), d-serine (d-Ser), and d-aspartate (d-Asp) have been reported to account for over 18% of total hydrolyzable amino acid (THAA) of bacterial peptidoglycan in the Chilean coastal sediments, and their percentages increase with the increase of water depths (up to over 26%) and sediment depth and ages (up to over 59%) (11). d-Enantiomers comprise 10 to 30% of Ala, Asp, Glu, and Ser in the high-molecular-weight fraction of oceanic DOM from the central Pacific Ocean, the Gulf of Mexico, and the North Sea (12), and represent >30% of total dissolved Ala, Asp, Glu, and Ser in the coastal waters of the North Atlantic (3). DAAs in the ocean are likely derived from diverse resources. While the vast majority of marine DAAs are released from bacterial peptidoglycan by protist grazing or viral and bacterial lysis (13, 14), some bacteria also secrete DAAs during their growth (3). However, how each DAA is metabolized by microorganisms to drive the recycling and mineralization of DAAs in the oceans is still largely unknown.
As a key component of bacterial peptidoglycan, d-Ala has the highest concentration of all DAAs in the total hydrolysable amino acids in natural seawater (15), and represents 44% and 21 to 40% of total dissolved Ala in deep waters of the eastern Arctic ocean (16) and North Atlantic waters (17), respectively. d-Ala is also the most abundant DAA in both high- and low-molecular-weight DOM fractions from the North Pacific Subtropical Gyre (18). The contents of free d-Ala, d-Glu, and d-Ser are in the range of 0.3 to 62 nmol/L in the seawater from Roskilde Fjord (13). Although the resting cells of some marine bacterial strains of Alphaproteobacteria and Gammaproteobacteria have been reported to have the activity to deaminate d-Ala into α-keto acids (19), our knowledge on the recycling of d-Ala in the ocean is still limited. It is still largely unknown what are the main microbial groups that utilize d-Ala in the ocean and how they metabolize d-Ala.
Pseudoalteromonas sp. strain CF6-2 is a Gram-negative bacterium isolated from a marine sediment (20). Strain CF6-2 can kill a variety of Gram-positive bacteria with diverse peptidoglycan chemotypes and utilize the released peptidoglycan-derived d-Ala and d-Glu for growth (21). In this study, we investigated the d-Ala catabolic pathways and identified the key enzymes involved in the catabolism of d-Ala in strain CF6-2. Unlike their terrestrial counterparts, which have DAA oxidoreductases/dehydrogenases or Ala racemase as the key enzymes in catabolizing d-Ala (22, 23), strain CF6-2 and other marine bacteria used d-Ala aminotransferase (TraA) as the essential enzyme for d-Ala catabolism, instead. Moreover, bioinformatic analysis suggested that marine bacteria possessing d-Ala aminotransferase genes are predominantly Gammaproteobacteria, which are widely distributed in global oceans. Therefore, Gammaproteobacteria is likely the dominant bacterial group to utilize d-Ala via d-Ala aminotransferase. These bacteria drive the recycling and mineralization of d-Ala in the oceans.
RESULTS
Identification of the key genes involved in d-Ala metabolism in strain CF6-2.
According to a previous study, the marine bacterium Pseudoalteromonas sp. strain CF6-2 can grow with 2 mM l/d-Ala and 50 mM glucose and prefers to utilize l-Ala when both enantiomers are present in the medium (21). When strain CF6-2 was cultured with different concentrations of l/d-Ala, the growth of strain CF6-2 increased with the concentration of l-Ala from 2 to 100 mM (Fig. 1A), which, however, reached the peak in the medium containing 20 mM d-Ala and significantly decreased in the media containing 50 to 100 mM d-Ala (Fig. 1B). This indicates that strain CF6-2 can utilize not only l-Ala but also d-Ala for growth and that d-Ala in the medium with a concentration higher than 20 mM has an inhibitory effect on the growth of strain CF6-2, thereby showing the toxicity of high concentration of d-Ala to strain CF6-2.
FIG 1.
(A and B) Growth of strain CF6-2 cultured with different concentrations of l-Ala (A) and d-Ala (B). Strain CF6-2 was cultured at 20°C in the media containing 50 mM glucose, 3.0% (wt/vol) synthetic sea salt, 0.2 M phosphate buffer (pH 8.0), and 0 to 100 mM d-Ala or l-Ala as indicated.
To probe the mechanism for d-Ala utilization of strain CF6-2, a global transcriptome analysis was performed on strain CF6-2 cultured with 2 mM l/d-Ala. Comparative transcriptomic data revealed three genes that are likely involved in d-Ala mechanism in strain CF6-2, a putative d-Ala aminotransferase gene traA (orf1756), a putative Ala racemase gene racA (orf0359), and a putative d-alanine-d-alanine ligase gene ligA (orf0208). The transcription level of traA in strain CF6-2 grown with d-Ala was markedly higher compared to l-Ala, while the transcription levels of racA and ligA were similar across the two test conditions (Table 1). Furthermore, quantitative proteome analysis showed that the abundance levels of TraA, RacA, and LigA were all upregulated (Table 2). Among these genes, ligA encodes a d-alanine-d-alanine ligase that is essential to synthetize peptidoglycan for cell wall construction whatever nitrogen source is used by a bacterium, and thus ligA is likely constitutively expressed and not specifically involved in d-Ala metabolism in strain CF6-2. Because the transcription and expression of both genes traA and racA were upregulated when strain CF6-2 was cultured with d-Ala (Tables 1 and 2) and both aminotransferase and racemase have been reported to be involved in DAA metabolism, it is possible that both genes traA and racA are involved in d-Ala metabolism in strain CF6-2. Therefore, we further investigated the functions of genes traA and racA in d-Ala metabolism in strain CF6-2.
TABLE 1.
Transcriptome analysis of the genes involved in d-Ala metabolism in strain CF6-2 cultured with l/d-Alaa
| Gene ID | Accession no. | Annotation | Fold change |
||
|---|---|---|---|---|---|
| d-Ala | l-Ala | d/l-Ala | |||
| orf1756 | MW435348 | d-Ala aminotransferase | 2.74 | 1.86 | 1.47 |
| orf3059 | MW435358 | Ala racemase | 3.51 | 3.21 | 1.09 |
| orf0208 | MW435368 | d-Alanine-d-alanine ligase | 1.39 | 1.11 | 1.26 |
The fold values were calculated by comparison to the values at 0 h.
TABLE 2.
Proteome analysis of the genes involved in d-Ala metabolism in strain CF6-2 cultured with d-Alaa
| Gene ID | Fold change |
|
|---|---|---|
| Exponential phase | Stationary phase | |
| orf1756 | 2.94 | 4.05 |
| orf3059 | 2.09 | 1.95 |
| orf0208 | 12.33 | 3.63 |
The fold values were calculated by comparison to the values at 0 h.
Analysis of the function of gene racA in d-Ala metabolism in strain CF6-2.
To analyze whether the racA gene of strain CF6-2 encodes a functional Ala racemase, we heterologously expressed racA in E. coli (Fig. 2A), and quantified the racemase activity of RacA using a circular dichroism (CD) method. Recombinant RacA racemized both d- and l-Ala (Fig. 2B and C), indicating that RacA catalyzes the reciprocal transformation between l-Ala and d-Ala. When strain CF6-2 was cultured with 2 mM d-Ala, RT-qPCR analysis showed that the transcription level of gene racA was upregulated at the exponential and stationary phases (Fig. 2D). Consistently, both proteome and Western blot analyses showed that the production of RacA was also upregulated at the exponential and stationary phases (Table 2 and Fig. 2D). Altogether, these results suggest that gene racA of strain CF6-2 encodes an Ala racemase, which is likely involved in d-Ala metabolism. Considering that the recombinant RacA of strain CF6-2 had the l-Ala racemization activity (Fig. 2C), it can be speculated that the function of RacA in strain CF6-2 cultured with l-Ala may be to convert l-Ala to d-Ala for peptidoglycan biosynthesis.
FIG 2.
Identification of the function of Ala racemase in d-Ala metabolism in strain CF6-2. (A) SDS-PAGE analysis of the purified recombinant RacA of strain CF6-2. (B) Analysis of the activity of recombinant RacA against l-Ala. (C) Analysis of the activity of recombinant RacA against d-Ala. (D) RT-qPCR analysis of the relative transcription levels of gene racA, and Western blot analysis of the expression level of RacA in strain CF6-2 cultured with d-Ala. The fold change was calculated by comparison to that at 0 h. The asterisks show statistical differences of the relative transcription levels of gene racA in the lag, exponential, and stationary phases (*, P < 0.05 [two-tailed t test]). (E) Growth of wild-type CF6-2 and ΔracA mutant strains cultured with different concentrations of d-Ala. (F) Growth of strain CF6-2-pEV and racA gene overexpression strain CF6-2-pEVracA cultured with different concentrations of d-Ala. The graph shows data from triplicate experiments (means ± the standard deviations [SD]).
To further evaluate the importance of racA in d-Ala metabolism in strain CF6-2, we constructed a knockout strain (ΔracA) and a racA overexpression strain, CF6-2-pEVracA, and investigated their growth with different concentrations of d-Ala. The results showed that there was little difference in the growth between the ΔracA mutant strain and the wild-type strain CF6-2 cultured with 2 to 20 mM d-Ala (Fig. 2E); however, the ability of strain CF6-2 to tolerate high concentrations (20 to 50 mM) of d-Ala was improved when gene racA was overexpressed (Fig. 2F). Thus, gene racA is not indispensable in d-Ala metabolism of strain CF6-2 in a low d-Ala concentration environment but may play a role in d-Ala detoxification in strain CF6-2 when it is in a high d-Ala concentration environment.
Analysis of the function of gene traA in d-Ala metabolism in strain CF6-2.
To understand the function of gene traA in d-Ala metabolism in strain CF6-2, gene traA was expressed in E. coli BL21(DE3) (Fig. 3A) and the transamination activities of the purified recombinant protein TraA toward d/l-Ala, d-Ala, and l-Ala were quantified using a spectrometric method. TraA showed high activities toward d/l-Ala and d-Ala, but almost no activity toward l-Ala (Fig. 3B), indicating that TraA is a specific d-Ala aminotransferase. The Km of TraA against d-Ala was 0.422 mM (Fig. 3C). The gene traA was significantly transcribed and translated when strain CF6-2 was cultured with 2 mM d-Ala (Fig. 3D), suggesting that traA is likely involved in d-Ala catabolism in strain CF6-2. We further evaluated the importance of TraA in d-Ala catabolism in strain CF6-2 by gene knockout. The ΔtraA mutant strain showed little growth in the medium with 2 mM d-Ala (Fig. 3E). When the traA gene was complemented into the ΔtraA strain, the complemented mutant strain restored the ability to utilize d-Ala and grew almost as well as the wild-type strain CF6-2 in the medium with 2 mM d-Ala (Fig. 3E). When the traA gene was overexpressed in the wild-type strain CF6-2, the growth of the strain in 20 to 50 mM d-Ala was better than that containing an empty vector, especially in 20 mM d-Ala (Fig. 3F), suggesting that the tolerance of strain CF6-2 to high concentration of d-Ala can be improved when traA is overexpressed.
FIG 3.
Identification of the function of d-Ala aminotransferase in d-Ala metabolism in strain CF6-2. (A) SDS-PAGE analysis of the purified recombinant TraA of strain CF6-2. (B) Analysis of the activity of the recombinant TraA against d/l-Ala, d-Ala, and l-Ala. One unit of enzyme activity was defined as the amount of enzyme that catalyzes Ala to generate 1 μmol pyruvate per h. (C) Michaelis-Menten curve of recombinant TraA against α-ketone glutaric acid. (D) RT-qPCR analysis of the relative transcription levels of gene traA and Western blot analysis of the protein expression level of TraA in strain CF6-2 cultured with 2 mM d-Ala. The asterisks show statistical differences of the relative transcription levels of gene traA in the lag, exponential and stationary phases (**, P < 0.01 [two-tailed t test]). (E) Growth of the ΔtraA mutant strain and the complementary strain ΔtraA-pEVtraA cultured with 2 mM d-Ala. (F) Growth of strain CF6-2-pEV and the traA gene overexpression strain CF6-2-pEVtraA cultured with different concentrations of d-Ala. The graph shows data from triplicate experiments (means ± the SD).
TraA catalyzes the transamination of d-Ala to generate another DAA and pyruvate. To investigate how the produced DAA is further metabolized by strain CF6-2, we analyzed the genome, transcriptome and proteome data. There are two genes that may be related to the metabolism of the DAA generated from the transamination of d-Ala catalyzed by TraA, a putative UDP-N-acetylmuramoylalanine-d-glutamate ligase gene (orf00203) and a putative UDP-N-acetylmuramoyl-l-alanyl-d-glutamate-2,6-diaminopimelate ligase gene (orf00200) (Table 3). The transcription levels of these two genes in strain CF6-2 cultured with d-Ala was higher than that with l-Ala, and the expression levels of these genes were all upregulated (Table 3). Because these two genes are both related to the biosynthesis of peptidoglycan for cell wall construction (24), the DAA generated from the transamination of d-Ala catalyzed by TraA is likely involved in the subsequent biosynthesis of peptidoglycan. In addition, although no other key genes related to DAA metabolism were found based on the analyses of genome, transcriptome and proteome data, it is not excluded that the DAA generated from the transamination of d-Ala catalyzed by TraA is further metabolized by other genes in strain CF6-2.
TABLE 3.
Transcriptomic and proteome analyses of genes involved in DAA metabolism in strain CF6-2 cultured with l/d-Alaa
| Gene annotation | Fold change |
||||
|---|---|---|---|---|---|
| Transcriptomic analysis |
Proteomic analysis (d-Ala) |
||||
| d-Ala | l-Ala | d/l-Ala | Exponential phase | Stationary phase | |
| UDP-N-acetylmuramoylalanine-d-glutamate ligase (orf00203) | 6.94 | 5.86 | 1.18 | 2.59 | 2.97 |
| UDP-N-acetylmuramoyl-l-alanyl-d-glutamate-2,6-diaminopimelate ligase (orf00200) | 4.96 | 3.86 | 1.28 | 5.27 | 2.73 |
The fold values were calculated by comparison to the values at 0 h.
Altogether, these results indicate that d-Ala transamination via TraA is an essential d-Ala catabolism pathway in strain CF6-2 when d-Ala is used as the nitrogen source.
A model for d-Ala metabolism in strain CF6-2.
Based on the above results, we concluded the mechanism of d-Ala catabolism in Pseudoalteromonas sp. strain CF6-2 as shown in Fig. 4. In the ocean, due to bacterial secretion (25) and bacterial peptidoglycan decomposition by protist grazing or viral and bacterial lysis (13, 14), free d-Ala is released, which is then utilized for nutrient by marine bacteria represented by strain CF6-2. When d-Ala molecules are transported into the cell of strain CF6-2, some d-Ala molecules are catalyzed by LigA to generate d-Ala-d-Ala to participate in the biosynthesis of peptidoglycan for cell wall construction. In the meantime, some d-Ala molecules are converted by RacA into l-Ala or by TraA to generate another DAA and pyruvate. The produced l-Ala, pyruvate, and other DAAs can be further metabolized to provide nutrient for growth or peptidoglycan biosynthesis. Among the three key enzymes, LigA is a constitutively expressed enzyme that is not specifically involved in d-Ala metabolism, TraA is an induced enzyme specific and essential for d-Ala catabolism, and RacA is an induced enzyme dispensable for d-Ala catabolism in strain CF6-2.
FIG 4.
d-Ala catabolism in marine bacterium Pseudoalteromonas sp. CF6-2. Key enzymes are marked with blue color. The solid black and gray arrows represent the essential and dispensable d-Ala catabolism pathways for the growth of strain CF6-2, respectively. d-Ala in the ocean can be from bacterial secretion or from bacterial peptidoglycan released by protist grazing or viral and bacterial lysis and utilized by marine bacteria such as strain CF6-2. When d-Ala is used as the nitrogen source, the expression of key genes traA and racA are induced. When transported into the bacterial cell, some d-Ala molecules are catalyzed by LigA to form d-Ala-d-Ala to participate in the biosynthesis of peptidoglycan for cell wall construction; in the meantime, some d-Ala molecules are converted by RacA into l-Ala or by TraA into DAA and pyruvate. The produced l-Ala, pyruvate, and DAA can be further metabolized to provide nutrient for the bacterial growth or to participate in peptidoglycan biosynthesis. TraA is essential and specific for d-Ala catabolism, but RacA is dispensable.
Investigation of the d-Ala-utilizing ability of other marine bacteria containing homologous racA and/or traA genes.
The above results indicate that racA and traA are two genes involved in d-Ala metabolism in strain CF6-2, for which traA is essential for growth. To investigate whether other marine bacteria use these genes for d-Ala utilization, we tested the d-Ala utilization ability of nine other marine bacterial strains (eight were Proteobacteria, and one was Bacteroidetes), seven of which contain racA and traA homologs, and the other two only have a racA homolog (Table 4). As shown in Fig. 5A, all seven strains containing traA and racA (racA+ traA+) could grow with 2 mM d-Ala, whereas none of the two strains without traA (racA+ ΔtraA) could grow, suggesting that d-Ala transamination via d-Ala aminotransferase is an essential pathway for these marine bacteria to metabolize d-Ala. To support this, we further detected the transcription levels of traA homologs in one Alphaproteobacteria strain (SM1341) and two Gammaproteobacteria strains (L1-12 and 34H) cultured with 2 mM d-Ala and the in vitro activity of the recombinant TraA-like proteins of these strains. The transcription levels of traA homologs, orf00392 and orf04366 in SM1341 and orf02526 in 34H, were significantly upregulated, and that of gene orf03825 in L1-12 was also upregulated, albeit to a lesser extent (Fig. 5B). Except that gene orf02526 from strain 34H was not successfully expressed in Escherichia coli, the recombinant proteins encoded by orf00392 and orf04366 of strain SM1341 and by orf03825 of strain L1-12 all demonstrated transamination activity against d-Ala (Fig. 5C and D). These results further indicate that the d-Ala aminotransferase gene is a specific gene for d-Ala catabolism in marine bacteria.
TABLE 4.
Key genes predicted to be involved in d-Ala metabolism in the investigated marine bacterial strains
| Strain and gene ID | Accession no. | Gene annotation |
|---|---|---|
| Oceanicella sp. SM1341 (Alphaproteobacteria) | ||
| orf00392 | MW435356 | d-Ala aminotransferase |
| orf04366 | MW435357 | d-Ala aminotransferase |
| orf02760 | MW435367 | Ala racemase |
| Vibrio lentus SM07-1 (Gammaproteobacteria) | ||
| orf01278 | MW435349 | Pyridoxal phosphate-dependent aminotransferase |
| orf02734 | MW435350 | Branched-chain-amino-acid aminotransferase |
| orf04285 | MW435359 | Ala racemase |
| Vibrio sp. SM13-9 (Gammaproteobacteria) | ||
| orf03928 | MW435351 | Branched-chain-amino-acid aminotransferase |
| orf04720 | MW435360 | Ala racemase |
| Paraglaciecola polaris L1-12 (Gammaproteobacteria) | ||
| orf03825 | MW435352 | d-Ala aminotransferase |
| orf03826 | MW435361 | Ala racemase |
| Paraglaciecola sp. 34H (Gammaproteobacteria) | ||
| orf02526 | MW435355 | d-Ala aminotransferase |
| orf02525 | MW435366 | Ala racemase |
| Vibrio pomeroyi SM11-13 (Gammaproteobacteria) | ||
| orf03912 | MW435353 | Branched-chain-amino-acid aminotransferase |
| orf03183 | MW435362 | Ala racemase |
| Vibrio gigantis SM12-8 (Gammaproteobacteria) | ||
| orf04626 | MW435354 | Branched-chain-amino-acid aminotransferase |
| orf04971 | MW435363 | Ala racemase |
| Parvularcula sp. SM1705 (Alphaproteobacteria) | ||
| orf02872 | MW435364 | Ala racemase |
| Winogradskyella thalassocola SA75 (Bacteroidetes) | ||
| orf00679 | MW435365 | Ala racemase |
FIG 5.
Analysis of the function of d-Ala aminotransferases in d-Ala metabolism in 9 marine bacterial strains. (A) Growth of marine bacterial strains cultured with 2 mM d-Ala. The black and red symbols represent the marine bacteria containing racA and traA homologs, and the gray symbols represent the marine bacteria only containing a racA homolog. (B) RT-qPCR analysis of the relative transcription levels of the traA genes in strains Oceanicella sp. SM1341, Paraglaciecola polaris L1-12, and Paraglaciecola sp. 34H cultured with d-Ala. The asterisks show statistical differences of the relative transcription levels of the traA genes in strains Oceanicella sp. SM1341, Paraglaciecola polaris L1-12, and Paraglaciecola sp. 34H in the lag and exponential phases (*, P < 0.05; **, P < 0.01 [two-tailed t test]). (C) SDS-PAGE analysis of the recombinant TraAs encoded by orf0392 (strain SM1341), orf4366 (strain L1-12), and orf3825 (strain 34H). (D) Analysis of the activity of the recombinant TraAs encoded by orf00392, orf04366, and orf03825 against d-Ala. The graph shows data from triplicate experiments (means ± the SD).
Diversity and distribution of marine bacteria containing d-Ala aminotransferase genes.
We further investigated the diversity and distribution of the traA gene in marine bacteria in order to explore the potential of d-Ala utilization. Since no e-value cutoff of d-Ala aminotransferase was reported, we analyzed the conserved amino acid residues at the active sites (K145, E177, and Y31) (26) of 10,483 d-Ala aminotransferase homologous sequences from the marine bacterial genomes in the Integrated Microbial Genome database of the Joint Genome Institute (i.e., the IMG/JGI database). Because the conservation of the three active site residues is higher than 95% when the e-value cutoff is e−50 (see Fig. S1 in the supplemental material), the IMG/JGI database and the Tara Oceans Microbiome database were probed for genomes of marine bacteria and metagenomes using the sequence of TraA of strain CF6-2 as the query with an e-value cutoff of e−50. In the IMG/JGI database, TraA-like sequences are found in the marine metagenome data set in different depths of seawater, sediments, deep subsurface, subseafloor crustal fluids, and hydrothermal vent samples, especially in seawater (Table 5). Furthermore, TraA-like sequences are found in the marine bacterial genomes in the IMG/JGI database, which mainly affiliate with the phylum Proteobacteria (88.42%), and to a lesser extent (11.58%) with other phyla (Firmicutes, 6.37%; unclassified bacteria, 4.21%) (Fig. 6A). TraA-like sequences are present in four classes of Proteobacteria, including Alphaproteobacteria (2.63%), Betaproteobacteria (8.42%), Epsilonproteobacteria (1.58%), and Gammaproteobacteria (75.79%). The predominance of TraA-like sequences in Gammaproteobacteria was also confirmed by probing its distribution in the Tara Oceans Microbiome database. According to the depth of sampling sites, Tara samples were divided into two groups, Tara-Surface (5 to 188 m) and Tara-Deep (250 to 1,000 m). We found 79.60 and 65.03% of TraA-like sequences from Tara-Surface and Tara-Deep, respectively, in Gammaproteobacteria, although the proportions of Gammaproteobacteria in the microbial community of the Tara-Surface (13.50%) and Tara-Deep (12.67%) samples are low (Fig. 6B), which further indicates that Gammaproteobacteria is the predominant group containing d-Ala aminotransferase. Thus, Gammaproteobacteria is likely the main bacterial group to utilize environmental d-Ala via d-Ala aminotransferase in the ocean.
TABLE 5.
Abundance of TraA homologs in the metagenomes from the IMG/JGI database
| Isolation samples | Depth (m) | Sequence no. | Ratio (%) |
|---|---|---|---|
| Surface seawater | 0–200 | 176 | 19.19 |
| Deep seawater | 200–4,020 | 226 | 24.65 |
| Seawater | 141 | 15.38 | |
| Sediment | 12 | 1.31 | |
| Deep subsurface | 470–9,177 | 15 | 1.64 |
| Subseafloor crustal fluids | 17 | 1.86 | |
| Hydrothermal vent | 1,672–2,514 | 14 | 1.53 |
| Others | 316 | 34.46 |
FIG 6.
Diversity and distribution of marine bacteria containing d-Ala aminotransferase genes. (A) Statistical analysis of marine bacteria containing TraA-like sequences from the marine bacterial genomes of the IMG/JGI database. (B) Relative percentage abundances of TraA-like sequences and microbial community composition of Tara samples retrieved from the Tara Oceans Microbiome databases. Tara samples were divided into two groups, Tara-Surface (5 to 188 m) and Tara-Deep (250 to 1,000 m). (C) Environmental distribution of predicted TraA-like sequences of Proteobacteria from the Tara Oceans Microbiome databases. Stations were plotted using Surfer version 12 (Golden Software LLC, USA) (38).
We further investigated the environmental distribution of these Proteobacteria TraA-like sequences in the Tara Oceans Microbiome databases. TraA-like-containing strains of Gammaproteobacteria are distributed at all sampling sites in the Tara Oceans Microbiome database, and TraA-like-containing strains of Betaproteobacteria and Alphaproteobacteria are also originally isolated from multiple habitats widely distributed in the ocean (Fig. 6C). These data indicate that bacteria utilizing d-Ala via d-Ala aminotransferase are distributed widely in the global oceans.
DISCUSSION
DAAs are important components of the dissolved organic matter (DOM) pool in the ocean (10). However, studies on marine DAA-utilizing organisms and their metabolism are rather less. There have been only a few reports on marine DAA-utilizing bacteria and their metabolism on DAAs. Kubota et al. isolated 28 DAA-utilizing bacterial strains from deep-sea sediments, which were phylogenetically assigned to Alphaproteobacteria, Gammaproteobacteria, and Bacilli (19). Yu et al. isolated seven DAA-utilizing bacterial strains from Arctic seawater and sediments (27). Both studies found that some bacterial strains have the ability to utilize multiple DAAs. Yu et al. also analyzed the metabolic pathways of the isolated bacteria on seven DAAs (d-Asp, d-Ser d-Leu, d-Met, d-Tyr, d-Thr, and d-Phe). In general, conversion of DAAs into α-keto acids is the main pathway in marine DAA-utilizing bacteria, which is performed by several key enzymes, including DAA oxidases/dehydrogenases, d-serine ammonia-lyases, d-serine ammonia-lyase DSD1, and DAA transaminases. In addition, conversion DAAs into LAAs is another pathway, which is performed by amino acid racemases (27).
As a key component of bacterial peptidoglycan, d-Ala is the most abundant DAA in the ocean. Although the resting cells of some strains of Alphaproteobacteria and Gammaproteobacteria have been shown to be able to deaminate d-Ala into α-keto acids (19), the catabolic pathways and the key enzymes involved in bacterial metabolization of d-Ala in the ocean is still elusive. In this study, we found that three enzymes are likely involved in d-Ala metabolism in marine bacterium Pseudoalteromonas sp. CF6-2, that is, d-Ala aminotransferase TraA, Ala racemase RacA, and d-alanine-d-alanine ligase LigA. LigA, which is likely constitutively expressed, generates peptide d-alanine-d-alanine with d-Ala to participate in the biosynthesis of peptidoglycan for cell wall construction. The expression of both TraA and RacA is induced in strain CF6-2 when d-Ala is utilized. TraA, which has activity toward only d-Ala, is a key enzyme specific for d-Ala catabolism and essential for the growth of strain CF6-2 cultured with d-Ala because the deletion of gene traA almost abolished the strain growth with d-Ala. However, RacA, which has activity toward both d-Ala and l-Ala, seems dispensable for d-Ala catabolism of strain CF6-2 because deletion of gene racA had little effect on the strain growth with d-Ala. These results suggest that, when d-Ala is used by strain CF6-2 for growth, the TraA pathway is the main and indispensable pathway for d-Ala catabolism and that the RacA pathway alone cannot provide enough nitrogen source for the bacterial growth. These results are also supported by our investigation on several other marine bacteria, which showed that those containing the d-Ala aminotransferase gene all have d-Ala-utilizing ability, but those without this gene do not have this ability.
The utilization of d-Ala in some terrestrial microorganisms have been investigated. Bacterial Ala racemases have been shown to play a key role in converting d-Ala to l-Ala on Earth (28). Racemization also plays a major role in the catabolism of d-Ala in the yeast Schizosaccharomyces pombe (23) and the archaea Methanococcus maripaludis (29). In addition, the d-amino acid dehydrogenase DadA of Pseudomonas aeruginosa has broad activity to a variety of DAAs, including d-Ala (30). However, d-Ala aminotransferase has not been found to play a key role in d-Ala catabolism in any terrestrial microorganism. In contrast, the results in this study show that marine bacteria, mainly Gammaproteobacteria, adopt another d-Ala metabolism pathway involving d-Ala aminotransferase as a key and specific enzyme.
Although it has been found that some strains of Alphaproteobacteria and Gammaproteobacteria can utilize d-Ala (19), our knowledge on the diversity of d-Ala-utilizing bacteria in the ocean is still limited. In this study, we investigated the diversity of bacteria containing the d-Ala aminotransferase gene in marine bacterial genomes and metagenomes in the IMG/JGI database and the Tara Oceans Microbiome database using TraA as the query sequence. The result showed that TraA-like sequences are predominantly present in Gammaproteobacteria, indicating that Gammaproteobacteria is likely the main bacterial group in the ocean to utilize environmental d-Ala via d-Ala aminotransferase. Thus, this study sheds light on the d-Ala-utilizing bacterial group in the ocean and its d-Ala-utilizing pathways, which is helpful in understanding the recycling and mineralization of d-Ala driven by bacteria in the global oceans.
Jorgensen and Middelboe (13) reported that the concentration of d-Ala in seawater is at the nM level. In this study, in order to observe significant bacterial growth and relevant gene expressions for probing the d-Ala metabolism, 2 mM d-Ala was used as the sole nitrogen source to cultivate marine bacteria, which is no doubt much higher than that in in-situ seawater. Moreover, in situ seawater likely contains various other nitrogen sources in addition to d-Ala. How marine bacteria metabolize d-Ala in in situ seawater with the presence of nM d-Ala and multiple other nitrogen sources still needs further investigation.
MATERIALS AND METHODS
Bacterial strains, medium, and materials.
Pseudoalteromonas sp. CF6-2 was originally isolated from a marine sediment sample from the South China Sea (20). Other bacterial strains used in this study were also previously isolated in our laboratory, including Oceanicella sp. SM1341 from South China Sea sediment, Paraglaciecola polaris L1-12 from Arctic seawater, Paraglaciecola sp. 34H from the intertidal soil of Goose Island, Parvularcula sp. SM1705 from South China Sea surface seawater, Winogradskyella thalassocola SA75 from Antarctic rotting algae, and Vibrio pomeroyi SM11-13, Vibrio gigantis SM12-8, Vibrio sp. SM13-9, and Vibrio lentus SM07-1 from Atlantic seawater. The knockout mutants of strain CF6-2 (ΔtraA and ΔracA), as well as their overexpression strains, CF6-2-pEVtraA and CF6-2-pEVracA, and the complemented mutant strain, ΔtraA-pEVtraA, were constructed according to the method described previously (31, 32). The plasmids, bacterial strains, and primers used in this study are listed in Tables S1 and S2 in the supplemental material. Utilization of d-Ala by strain CF6-2 and the other strains shown in Table 4 was detected with an interval of 4 h by automatically recording the growth as the optical density at 600 nm (OD600) of each strain cultured at 20°C on a FP-1000-C automated microbiology growth curve analysis system (Finland). In this experiment, 1% (vol/vol) cell suspension of each strain (OD600 ≈ 0.8) was inoculated into the defined medium containing 50 mM glucose (Sinopharm, China), 3.0% (wt/vol) synthetic sea salt (Sigma-Aldrich, USA), 0.2 M phosphate buffer (pH 8.0), and 2 to 100 mM d-Ala or l-Ala (Solarbio, China) wherever appropriate. Rabbit polyclonal antibodies for Western blot analysis were prepared by the Beijing Genomics Institute, China.
Genome analysis.
Functional annotation of the predicted genes in the previously sequenced genomes of strain CF6-2 and the nine strains listed in Table 4 was carried out using BLASTP with the NCBI nonredundant protein database and the Kyoto Encyclopedia of Genes and Genomes (KEGG) protein database (33).
Transcriptome analysis.
Strain CF6-2 was cultured in 2216E medium at 20°C to an OD600 of 0.8. The cells in the culture were collected by centrifugation at 4,000 × g for 10 min at 4°C, washed three times with 3% (wt/vol) sea salt solution, and then inoculated into the defined medium with 2 mM d-Ala. Samples were taken at the lag phase and the exponential phase. Samples were collected by centrifugation at 4,000 × g and 4°C for 10 min. The resulting pellets were frozen in liquid nitrogen and stored at −80°C. Transcriptome sequencing and analysis were performed by WHBioacme Technology Co., Ltd, China. Significant differences were indicated by P values of <0.05 and an absolute fold change threshold of >2.0.
Proteomic analysis.
Bacterial cells of strain CF6-2 cultured with 2 mM d-Ala were collected at the lag phase and the exponential phase. Tandem Mass Tag (TMT)-based quantitative proteomic analysis was performed by Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China. LC-MS/MS/MS analysis was performed using an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Scientific, Rockford, IL) coupled online to an Easy-nLC 1200 in the data-dependent mode. The database search was performed for all raw MS files using the software MaxQuant (v1.6.0.16). The parameters used for the database search were set up as follows: type of search, MS3; type of isobaric labels,10-plex TMT; mass tolerance for precursor ions, 20 ppm for the first search and 4.5 ppm for the main search; mass tolerance for fragments ions, 20 ppm; minimum score for unmodified peptides, 15; and minimum score for modified peptides, 40. Variable modifications include N-terminal acetylation and methionine oxidation; the fix modification includes cysteine carbamidomethylation. The false discovery rates for both peptide and protein identifications were set to 0.01. Default values were used for all other parameters.
Real-time qPCR analysis.
Bacterial cells cultured with 2 mM d-Ala were collected at the lag phase, the exponential phase, and the stationary phase. The bacterial cells were treated with 66 nmol of lysozyme at 37°C for 30 min to lyse the cell walls, and then total RNA was extracted with an RNeasy minikit (Qiagen, Valencia, CA). Reverse transcription was performed using the PrimeScript RT reagent kit with gDNA Eraser (Perfect Real Time; TaKaRa, Japan). The qPCR was performed on the LightCycler 480 (Roche, Switzerland). The relative expression level was indicated as fold change which was calculated using the LightCycler 480 software according to the 2–ΔΔCT method. Each sample for qPCR was performed in triplicate and a mean value and standard deviation were calculated. The recA gene was used as the reference gene.
Western blot analysis.
Western blotting was performed as described previously (35). The blots were probed for TraA and RacA using a TraA polyclonal antibody and a RacA polyclonal antibody, respectively. After treatment with a horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit ab6721; Abcam), the blots were imaged in chemiluminescent solution (GE Healthcare/Amersham ECL Prime Western blotting detection reagent, catalog no. RPN2232) on a myECL imager (Thermo Scientific).
Gene cloning, protein expression, and purification.
Genes were cloned from bacterial genome DNA via PCR, including the traA and racA genes from the genome of strain CF6-2 and d-Ala aminotransferase genes from the genomes of strains SM1341, L1-12, and 34H. All genes were overexpressed in E. coli BL21(DE3) cells using a pET-22b vector that contains a His tag for protein purification. Recombinant E. coli strains were cultured at 20°C for 18 h with 0.4 mM isopropyl-β-d-thiogalactopyranoside (IPTG). Recombinant proteins were purified with Ni2+-NTA resin (Qiagen, Germany), followed by desalination on a PD-10 desalting column (GE Healthcare, USA).
Enzyme assays.
The activity of Ala racemase was assayed by the CD method (36). The reaction mixture (0.5 mL) containing 10 mM NaHCO3-NaOH buffer (pH 10.5), 100 mM d-Ala/l-Ala, and 1.6 nmol of purified enzyme was incubated at 20°C for 10 min. The same reaction mixture without the purified enzyme was used as a control. l-Ala and d-Ala were used as calibration standards. Measurement of CD spectra was carried out in a 0.1-cm-path length cell on a JASCO J-1500 spectrometer (Japan). The scan rate and bandwidth were set to 500 nm/min and 1.0 nm, respectively. Spectra were recorded between 190 and 250 nm by 0.5-nm carving and were averaged from three scans. The activity of d-Ala aminotransferase was measured by the color reaction between the product pyruvate and 2,4-dinitrophenylhydrazine (37). The reaction mixture (500 μL) containing 100 mM phosphate-buffered saline (pH 7.4), 100 mM l/d/ld-Ala, 2 mM α-ketoglutaric acid, and 50 μL of enzyme was incubated at 20°C for 30 min. After the reaction, 1 mM 2, 4-dinitrophenylhydrazine was added to the reaction mixture, followed by further incubation at 20°C for 20 min. Then, 0.4 M NaOH was added to the mixture, and the mixture was incubated at 20°C for 10 min. Next, the absorbance of the mixture at 520 nm was measured against a blank that contained all of the components except for the purified enzyme. Pyruvate was used as a calibration standard.
Data availability.
All the RNA-seq read data have been deposited in the NCBI Sequence Read Archive (SRA) under project accession number PRJNA688555. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (34) partner repository with the data set identifier PXD023403.
ACKNOWLEDGMENTS
We thank Jingyao Qu, Jing Zhu, and Zhifeng Li from the State Key Laboratory of Microbial Technology of Shandong University for help and guidance in DLS.
This study was supported by the National Science Foundation of China (U1706207, 31630012, 91851205, U2006205, 31800107, and 42076229), the National Key Research and Development Program of China (2018YFC1406700 and 2018YFC0310704), the Fundamental Research Funds for the Central Universities (202172002), Major Scientific and Technological Innovation Project (MSTIP) of Shandong Province (2019JZZY010817), and the Program of Shandong for Taishan Scholars (tspd20181203).
We declare there are no conflicts of interest.
Footnotes
Supplemental material is available online only.
Contributor Information
Jun-Mei Ding, Email: djm3417@163.com.
Xiu-Lan Chen, Email: cxl0423@sdu.edu.cn.
Laura Villanueva, Royal Netherlands Institute for Sea Research.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Tables S1 and S2, Fig. S1. Download aem.02219-21-s0001.pdf, PDF file, 0.2 MB (229.7KB, pdf)
Data Availability Statement
All the RNA-seq read data have been deposited in the NCBI Sequence Read Archive (SRA) under project accession number PRJNA688555. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (34) partner repository with the data set identifier PXD023403.