Ni Uptake and Limitation in Marine Synechococcus Strains

Ni accumulation and utilization were studied in two strains of marine Synechococcus, isolated from both coastal (CC9311; clade I) and open-ocean (WH8102; clade III) environments, for which complete genome sequences are available. Both strains have genes encoding an Ni-containing urease and when grown on urea without Ni become Ni-N colimited. The Ni requirements of these strains also depend upon the genomic complement of genes encoding superoxide dismutase (SOD). WH8102, with a gene encoding only an Ni-SOD, has a novel obligate requirement for Ni, regardless of the N source. Reduced SOD activity in Ni-depleted cultures of WH8102 supports the link of this strain's Ni requirement to Ni-SOD. The genome of CC9311 contains a gene for a Cu/Zn-SOD in addition to a predicted pair of Ni-SODs, yet this strain cannot grow without Ni on NO₃⁻ and can grow only slowly on NH₄⁺ without Ni, implying that the Cu/Zn-SOD cannot completely replace Ni-SOD in marine cyanobacteria. CC9311 does have a greater tolerance for Ni starvation. Both strains increase their Ni uptake capabilities and actively bioconcentrate Ni in response to decreasing extracellular and intracellular Ni. The changes in Ni uptake rates were more pronounced in WH8102 than in CC9311 and for growth on urea or nitrate than for growth on ammonia. These results, combined with an analysis of fully sequenced marine cyanobacterial genomes, suggest that the growth of many marine Synechococcus and all Prochlorococcus strains is dependent upon Ni.     

Identification of glycine betaine as compatible solute in Synechococcus sp. WH8102 and characterization of its N-methyltransferase genes involved in betaine synthesis

Biosynthesis of glycine betaine from simple carbon sources as compatible solute is rare among aerobic heterotrophic eubacteria, and appears to be almost exclusive to the non-halophilic and slightly halophilic phototrophic cyanobacteria. Although Synechococcus sp. WH8102 (CCMP2370), a unicellular marine cyanobacterium, could grow up to additional 2.5% (w/v) NaCl in SN medium, natural abundance ¹³C nuclear magnetic resonance spectroscopy identified glycine betaine as its major compatible solute. Intracellular glycine betaine concentrations were dependent on the osmolarity of the growth medium over the range up to additional 2% NaCl in SN medium, increasing from 6.8 ± 1.5 to 62.3 ± 5.5 mg/g dw. The ORFs SYNW1914 and SYNW1913 from Synechococcus sp. WH8102 were found as the homologous genes coding for glycine sarcosine N-methyltransferase and sarcosine dimethylglycine N-methyltransferase, heterologously over-expressed respectively as soluble fraction in Escherichia coli BL21(DE3)pLysS and purified by Ni-NTA His•bind resins. Their substrate specificities and the values of the kinetic parameters were determined by TLC and ¹H NMR spectroscopy. RT-PCR analysis revealed that the two ORFs were both transcribed in cells of Synechococcus sp. WH8102 growing in SN medium without additional NaCl, which confirmed the pathway of de novo synthesizing betaine from glycine existing in these marine cyanobacteria.     

DIFFERENT PHYSIOLOGICAL RESPONSES OF FOUR MARINE SYNECHOCOCCUS STRAINS (CYANOPHYCEAE) TO NICKEL STARVATION UNDER IRON‐REPLETE AND IRON‐DEPLETE CONDITIONS1

Synechococcus species are important primary producers in coastal and open‐ocean ecosystems. When nitrate was provided as the sole nitrogen source, nickel starvation inhibited the growth of strains WH8102 and WH7803, while it had little effect on two euryhaline strains, WH5701 and PCC 7002. Nickel was required for the acclimation of Synechococcus WH7803 to low iron and high light. In WH8102 and WH7803, nickel starvation decreased the linear electron transport activity, slowed down Q_A reoxidation, but increased the connectivity factor between individual photosynthetic units. Under such conditions, the reduction of their intersystem electron transport chains was expected to increase, and their cyclic electron transport around PSI would be favored. Nickel starvation decreased the total superoxide dismutase (SOD) activity of WH8102 and WH7803 by 30% and 15% of the control, respectively. The protein‐bound ⁶³Ni of the oceanic strain WH8102 comigrated with SOD activity on nondenaturing gels and thus provided additional evidence for the existence of active NiSOD in Synechococcus WH8102. In WH7803, it seems likely that nickel starvation affected other metabolic pathways and thus indirectly affected the total SOD activity.     

Comparative membrane proteomics reveal contrasting adaptation strategies for coastal and oceanic marine Synechococcus cyanobacteria

Marine cyanobacteria genus Synechococcus are among the most abundant and widespread primary producers in the open ocean. Synechococcus strains belonging to different clades have adapted distinct strategies for growth and survival across a range of marine conditions. Clades I and IV are prevalent in colder, mesotrophic, coastal waters, while clades II and III prefer warm, oligotrophic open oceans. To gain insight into the cellular resources these unicellular organisms invest in adaptation strategies we performed shotgun membrane proteomics of four Synechococcus spp. strains namely CC9311 (clade I), CC9605 (clade II), WH8102 (clade III) and CC9902 (clade IV). Comparative membrane proteomes analysis demonstrated that CC9902 and WH8102 showed high resource allocation for phosphate uptake, accounting for 44% and 38% of overall transporter protein expression of the species. WH8102 showed high expression of the iron uptake ATP-binding cassette binding protein FutA, suggesting that a high binding affinity for iron is possibly a key adaptation strategy for some strains in oligotrophic ocean environments. One protein annotated as a phosphatase 2c (Sync_2505 and Syncc9902_0387) was highly expressed in the coastal mesotrophic strains CC9311 and CC9902, constituting 14%–16% of total membrane protein, indicating a vital, but undefined function, for strains living in temperate mesotrophic environments.     

Computational inference and experimental validation of the nitrogen assimilation regulatory network in cyanobacterium Synechococcus sp. WH 8102

Deciphering the regulatory networks encoded in the genome of an organism represents one of the most interesting and challenging tasks in the post-genome sequencing era. As an example of this problem, we have predicted a detailed model for the nitrogen assimilation network in cyanobacterium Synechococcus sp. WH 8102 (WH8102) using a computational protocol based on comparative genomics analysis and mining experimental data from related organisms that are relatively well studied. This computational model is in excellent agreement with the microarray gene expression data collected under ammonium-rich versus nitrate-rich growth conditions, suggesting that our computational protocol is capable of predicting biological pathways/networks with high accuracy. We then refined the computational model using the microarray data, and proposed a new model for the nitrogen assimilation network in WH8102. An intriguing discovery from this study is that nitrogen assimilation affects the expression of many genes involved in photosynthesis, suggesting a tight coordination between nitrogen assimilation and photosynthesis processes. Moreover, for some of these genes, this coordination is probably mediated by NtcA through the canonical NtcA promoters in their regulatory regions.     

Structure of Compositionally Simple Lipopolysaccharide from Marine Synechococcus

Lipopolysaccharide (LPS) is the first defense against changing environmental factors for many bacteria. Here, we report the first structure of the LPS from cyanobacteria based on two strains of marine Synechococcus, WH8102 and CC9311. While enteric LPS contains some of the most complex carbohydrate residues in nature, the full-length versions of these cyanobacterial LPSs have neither heptose nor 3-deoxy-d-manno-octulosonic acid (Kdo) but instead 4-linked glucose as their main saccharide component, with low levels of glucosamine and galacturonic acid also present. Matrix-assisted laser desorption ionization mass spectrometry of the intact minimal core LPS reveals triacylated and tetraacylated structures having a heterogeneous mix of both hydroxylated and nonhydroxylated fatty acids connected to the diglucosamine backbone and a predominantly glucose outer core-like region for both strains. WH8102 incorporated rhamnose in this region as well, contributing to differences in sugar composition and possibly nutritional differences between the strains. In contrast to enteric lipid A, which can be liberated from LPS by mild acid hydrolysis, lipid A from these organisms could be produced by only two novel procedures: triethylamine-assisted periodate oxidation and acetolysis. The lipid A contains odd-chain hydroxylated fatty acids, lacks phosphate, and contains a single galacturonic acid. The LPS lacks any limulus amoebocyte lysate gelation activity. The highly simplified nature of LPSs from these organisms leads us to believe that they may represent either a primordial structure or an adaptation to the relatively higher salt and potentially growth-limiting phosphate levels in marine environments.Lipopolysaccharide (LPS) in the outer membrane layer is known to be the first line of defense against environmental factors in many gram-negative organisms, preventing lysis by complement, antimicrobial peptides and detergents (17, 21, 47). In proteobacteria, 3-deoxy-d-manno-octulosonic acid (Kdo), heptose, and phosphate are key parts of the conserved inner core of the LPS which connects the less-well-conserved outer core and sometimes an attached polysaccharide to the lipid A anchor. Why heptose is so well conserved is a mystery, but the prevalence of Kdo and phosphate may be related to the charge which they impart to the outer membrane and to their ability to bind divalent cations. The Kdo-phosphate metal binding center is capable of binding calcium with a dissociation constant (K_d) of 12 to 13 μM (28). This high-affinity binding of divalent cations is known to be necessary for the low permeability of LPS bilayers to some antibiotics (32), and it has been hypothesized that divalent cation cross-bridges may link LPS molecules on the bacterial cell surfaces of enterobacteria into a giant complex with very low membrane permeability (16).Though the LPSs of many proteobacteria are well characterized, the LPSs from cyanobacteria are much less studied. The cell envelopes of cyanobacteria resemble those of gram-negative bacteria structurally, consisting of a cytoplasmic membrane, a peptidoglycan layer, an outer membrane containing LPS, and sometimes additional structures (9, 14). Previous chemical analyses have shown the LPS of some cyanobacteria to be devoid of phosphate, Kdo, and heptose (11, 12, 42, 43). Given the lack of Kdo in these organisms as well as the fact that the lability of the Kdo-glucosamine ketosidic linkage allows for the mild acid hydrolysis of LPS to lipid A, it is perhaps not surprising that many attempts at hydrolysis of cyanobacterial LPS to lipid A have failed (for an example, see reference 29).Within the cyanobacteria, the genus Synechococcus represents a polyphyletic group of unicellular morphotypes. Synechococcus cells are found in both freshwater and marine environments. Organisms from group A Synechococcus and its sister taxon Prochlorococcus are extremely important primary producers in marine environments, with multiple “clades” similar to “species” described for other bacteria, dominating in different environments (3, 22). Unlike enterobacteria, which must frequently contend with an onslaught of host factors, members of the Synechococcus face grazing by protists and bacteriophages as their primary survival challenges.The genome of Synechococcus sp. strain CC9311 has been shown to be devoid of the genes for Kdo biosynthesis, while strain WH8102 has several putative genes for Kdo biosynthesis (18, 20). This suggests that the LPS of cyanobacteria could be significantly different from that of enteric bacteria and could show species/strain variation as well. A comparison of the structures of LPS from cyanobacteria and enterobacteria would afford a unique opportunity to understand which elements of LPS structure are essential to bacterial survival and which are adaptations to the environment in which the bacteria live. To further this understanding, we present here an analysis of the LPS structure from two strains of marine Synechococcus: an open-ocean-dwelling strain having the putative genes for Kdo biosynthesis (strain WH8102; clade III) and a coastal strain lacking these genes (strain CC9311; clade I). We further present two novel methods for producing lipid A from bacteria lacking the labile Kdo ketosidic linkage.     

Clade-Specific 16S Ribosomal DNA Oligonucleotides Reveal the Predominance of a Single Marine Synechococcus Clade throughout a Stratified Water Column in the Red Sea

Phylogenetic relationships among members of the marine Synechococcus genus were determined following sequencing of the 16S ribosomal DNA (rDNA) from 31 novel cultured isolates from the Red Sea and several other oceanic environments. This revealed a large genetic diversity within the marine Synechococcus cluster consistent with earlier work but also identified three novel clades not previously recognized. Phylogenetic analyses showed one clade, containing halotolerant isolates lacking phycoerythrin (PE) and including strains capable, or not, of utilizing nitrate as the sole N source, which clustered within the MC-A (Synechococcus subcluster 5.1) lineage. Two copies of the 16S rRNA gene are present in marine Synechococcus genomes, and cloning and sequencing of these copies from Synechococcus sp. strain WH 7803 and genomic information from Synechococcus sp. strain WH 8102 reveal these to be identical. Based on the 16S rDNA sequence information, clade-specific oligonucleotides for the marine Synechococcus genus were designed and their specificity was optimized. Using dot blot hybridization technology, these probes were used to determine the in situ community structure of marine Synechococcus populations in the Red Sea at the time of a Synechococcus maximum during April 1999. A predominance of genotypes representative of a single clade was found, and these genotypes were common among strains isolated into culture. Conversely, strains lacking PE, which were also relatively easily isolated into culture, represented only a minor component of the Synechococcus population. Genotypes corresponding to well-studied laboratory strains also appeared to be poorly represented in this stratified water column in the Red Sea.     

Cyanophycin Production in a Phycoerythrin-Containing Marine Synechococcus Strain of Unusual Phylogenetic Affinity

Thirty-two strains of phycoerythrin-containing marine picocyanobacteria were screened for the capacity to produce cyanophycin, a nitrogen storage compound synthesized by some, but not all, cyanobacteria. We found that one of these strains, Synechococcus sp. strain G2.1 from the Arabian Sea, was able to synthesize cyanophycin. The cyanophycin extracted from the cells was composed of roughly equimolar amounts of arginine and aspartate (29 and 35 mol%, respectively), as well as a small amount of glutamate (15 mol%). Phylogenetic analysis, based on partial 16S ribosomal DNA (rDNA) sequence data, showed that Synechococcus sp. strain G2.1 formed a well-supported clade with several strains of filamentous cyanobacteria. It was not closely related to several other well-studied marine picocyanobacteria, including Synechococcus strains PCC7002, WH7805, and WH8018 and Prochlorococcus sp. strain MIT9312. This is the first report of cyanophycin production in a phycoerythrin-containing strain of marine or halotolerant Synechococcus, and its discovery highlights the diversity of this ecologically important functional group.     

Examination of Genetic Relatedness of Marine Synechococcus spp. by Using Restriction Fragment Length Polymorphisms

The relatedness of several marine Synechococcus spp. was estimated by DNA hybridization. Strains isolated from various geographical locations and representing a diversity of DNA base compositions and phycobiliprotein profiles were compared by restriction fragment length polymorphisms for a number of genes. DNAs from two marine red algae and a cryptomonad alga (which exhibit a phycobiliprotein composition similar to that of the marine Synechococcus spp.) and Synechococcus strain PCC6301 (Anacystis nidulans) were also included in the comparison. Strains WH8008, WH8018, and WH7805 were shown to be very similar to one another, as were strains WH7802 and WH7803. Strains WH8110 and WH5701 were clearly unrelated to any of the other strains, and no marine Synechococcus isolate showed any similarity to the freshwater Synechococcus strain PCC6301 or the eucaryotic algae. The method is relatively straightforward and sensitive and uses a variety of basic molecular biology techniques. Its utility in ascertaining the genetic relatedness and diversity of marine Synechococcus spp. and possible extension to field studies are discussed.     

Selection and Characterization of Cyanophage Resistance in Marine Synechococcus Strains

Marine viruses are an important component of the microbial food web, influencing microbial diversity and contributing to bacterial mortality rates. Resistance to cooccurring cyanophages has been reported for natural communities of Synechococcus spp.; however, little is known about the nature of this resistance. This study examined the patterns of infectivity among cyanophage isolates and unicellular marine cyanobacteria (Synechococcus spp.). We selected for phage-resistant Synechococcus mutants, examined the mechanisms of phage resistance, and determined the extent of cross-resistance to other phages. Four strains of Synechococcus spp. (WH7803, WH8018, WH8012, and WH8101) and 32 previously isolated cyanomyophages were used to select for phage resistance. Phage-resistant Synechococcus mutants were recovered from 50 of the 101 susceptible phage-host pairs, and 23 of these strains were further characterized. Adsorption kinetic assays indicate that resistance is likely due to changes in host receptor sites that limit viral attachment. Our results also suggest that receptor mutations conferring this resistance are diverse. Nevertheless, selection for resistance to one phage frequently resulted in cross-resistance to other phages. On average, phage-resistant Synechococcus strains became resistant to eight other cyanophages; however, there was no significant correlation between the genetic similarity of the phages (based on g20 sequences) and cross-resistance. Likewise, host Synechococcus DNA-dependent RNA polymerase (rpoC1) genotypes could not be used to predict sensitivities to phages. The potential for the rapid evolution of multiple phage resistance may influence the population dynamics and diversity of both Synechococcus and cyanophages in marine waters.     

Swimming Motility Mutants of Marine Synechococcus Affected in Production and Localization of the S-Layer Protein SwmA

The S-layer protein SwmA is required for nonflagellar swimming in marine Synechococcus. An analysis of mutations in seven genes at two loci in the Synechococcus sp. strain WH8102 genome indicates that a multicomponent transporter and glycosyltransferases are required for the production and proper localization of SwmA.The mechanism of nonflagellar motility by which certain strains of marine Synechococcus swim in the absence of any extracellular organelle remains mysterious. The cell surface itself is predicted to produce thrust (11), and to date, two cell surface proteins required for swimming have been characterized (7, 14). SwmA is a 130-kDa glycoprotein that forms a paracrystalline surface layer (S-layer) (16). Whether the S-layer plays a direct role in motility or a more indirect role, e.g., being required for the proper placement and functioning of other components of the motility apparatus, remains unclear. SwmB is a highly repetitive, 1.12-MDa protein which is also required for motility and is similarly localized near the cell surface where it is arranged in a punctate manner (14).Transposon mutagenesis identified three separate chromosomal regions required for swimming motility in Synechococcus sp. strain WH8102 (15). In addition to the genes coding for SwmA and SwmB, two separate multicomponent ABC transporter genes, several putative glycosyltransferase genes, and various conserved and hypothetical genes of unknown function comprise the remaining genes present in these motility loci (15). We show here that mutations in several of these open reading frames (ORFs; SYNW0079, SYNW0087 to SYNW0089, and SYNW0192 to SYNW0195) affect the production and cellular localization of SwmA, and in the case of SYNW0087 and SYNW0195, that of a 70-kDa outer membrane protein (OMP).     

Influence of N and Ni supply on nitrogen metabolism and urease activity in rice (Oryza sativa L.)

Nickel is considered to be an essential micronutrient in plants because of its role in the metalloenzyme urease. In order to characterize the metabolic consequences of Ni deprivation, the significance of Ni supply for growth and N metabolism of rice plants grown with either NH4NO3 or urea as sole N source was evaluated. Growth of plants receiving NH4NO3 was not affected by the Ni status, and neither were the activities of arginase and glutamine synthetase. However, urease activity was not detectable in leaves of low-Ni plants, which in conjunction with arginase action, led to the accumulation of urea in plants grown with NH4NO3. Amino acid contents and mineral nutrient status (except Ni) were not affected by the Ni treatment.Urea-grown Ni-deprived plants showed reduced growth and accumulated large amounts of urea owing to the lack of urease activity. These plants were further characterized by low amino acid contents indicating impaired usage of the N supplied. They also exhibited reduced levels of the urea precursor arginine, which is merely attributed to the overall N economy in these plant. When urea-grown plants were supplied with 0.5 mmol m^-3 Ni in the nutrient solution, the dry weight and the amino acid N contents were increased at the expense of the urea contents, indicating efficient use of urea N in Ni-supplemented plants.A critical Ni concentration in the shoot regarding dry matter production of NH4NO3-grown plants could not be deduced, while 25 g Ni kg^-1 DW is certainly inadequate for urea-grown plants. This suggests that the Ni requirement strongly depends on the N source employed.Keywords: Amino acids, ornithine cycle, Ni supply, rice, urea, urease activity.      

Swimming Marine Synechococcus Strains with Widely Different Photosynthetic Pigment Ratios Form a Monophyletic Group

Unicellular marine cyanobacteria are ubiquitous in both coastal and oligotrophic regimes. The contribution of these organisms to primary production and nutrient cycling is substantial on a global scale. Natural populations of marine Synechococcus strains include multiple genetic lineages, but the link, if any, between unique phenotypic traits and specific genetic groups is still not understood. We studied the genetic diversity (as determined by the DNA-dependent RNA polymerase rpoC1 gene sequence) of a set of marine Synechococcus isolates that are able to swim. Our results show that these isolates form a monophyletic group. This finding represents the first example of correspondence between a physiological trait and a phylogenetic group in marine Synechococcus. In contrast, the phycourobilin (PUB)/phycoerythrobilin (PEB) pigment ratios of members of the motile clade varied considerably. An isolate obtained from the California Current (strain CC9703) displayed a pigment signature identical to that of nonmotile strain WH7803, which is considered a model for low-PUB/PEB-ratio strains, whereas several motile strains had higher PUB/PEB ratios than strain WH8103, which is considered a model for high-PUB/PEB-ratio strains. These findings indicate that the PUB/PEB pigment ratio is not a useful characteristic for defining phylogenetic groups of marine Synechococcus strains.     

Effects of iron on the growth and minimal fluorescence yield of three marine Synechococcus strains (Cyanophyceae)

Two coastal Synechococcus stains PCC 7002 and CC9311 and one oceanic strain WH8102 were cultured with 4–1000 nM Fe in Aquil medium. Compared with those under iron‐replete conditions, their growth rates were significantly decreased by 59% for WH8102 at 15 nM Fe, by 37% for CC9311 at 15 nM Fe and by 57% for PCC 7002 at 4 nM Fe. Among these three strains, PCC 7002 was the most tolerant to iron limitation while WH8102 was the most sensitive to iron limitation. For each strain under the same iron concentration, the growth rates calculated from the minimal fluorescence yield and cell concentration showed no significant difference. The linear correlation was established between the minimal fluorescence yield and cell concentration although the minimal fluorescence yield per cell varied depending on the strains and iron levels. Under iron‐replete conditions, the minimal fluorescence yield per cell was 100‐fold higher for the phycoerythrin‐lacking strain PCC 7002 than two phycoerythrin‐containing strains WH8102 and CC9311. Under iron‐deplete conditions, it was increased respectively by 128% and 7% for WH8102 and CC9311 but was decreased by 30% for PCC 7002. Furthermore, the minimal fluorescence yield per cell for PCC 7002 and CC9311 showed little difference throughout the light and dark diel cycle. However, it was significantly higher for WH8102 in the daytime than in the dark.     

Photophysiological and Photosynthetic Complex Changes during Iron Starvation in Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942

Iron is an essential component in many protein complexes involved in photosynthesis, but environmental iron availability is often low as oxidized forms of iron are insoluble in water. To adjust to low environmental iron levels, cyanobacteria undergo numerous changes to balance their iron budget and mitigate the physiological effects of iron depletion. We investigated changes in key protein abundances and photophysiological parameters in the model cyanobacteria Synechococcus PCC 7942 and Synechocystis PCC 6803 over a 120 hour time course of iron deprivation. The iron stress induced protein (IsiA) accumulated to high levels within 48 h of the onset of iron deprivation, reaching a molar ratio of ∼42 IsiA : Photosystem I in Synechococcus PCC 7942 and ∼12 IsiA : Photosystem I in Synechocystis PCC 6803. Concomitantly the iron-rich complexes Cytochrome b₆f and Photosystem I declined in abundance, leading to a decrease in the Photosystem I : Photosystem II ratio. Chlorophyll fluorescence analyses showed a drop in electron transport per Photosystem II in Synechococcus, but not in Synechocystis after iron depletion. We found no evidence that the accumulated IsiA contributes to light capture by Photosystem II complexes.     

A Suppression Subtractive Hybridization Approach Reveals Niche-Specific Genes That May Be Involved in Predator Avoidance in Marine Synechococcus Isolates

Picocyanobacteria of the genus Synechococcus are important contributors to marine primary production and are ubiquitous in the world's oceans. This genus is genetically diverse, and at least 10 discrete lineages or clades have been identified phylogenetically. However, little if anything is known about the genetic attributes which characterize particular lineages or are unique to specific strains. Here, we used a suppression subtractive hybridization (SSH) approach to identify strain- and clade-specific genes in two well-characterized laboratory strains, Synechococcus sp. strain WH8103 (clade III) and Synechococcus sp. strain WH7803 (clade V). Among the genes that were identified as potentially unique to each strain were genes encoding proteins that may be involved in specific predator avoidance, including a glycosyltransferase in strain WH8103 and a permease component of an ABC-type polysaccharide/polyol phosphate export system in WH7803. During this work the genome of one of these strains, WH7803, became available. This allowed assessment of the number of false-positive sequences (i.e., sequences present in the tester genome) present among the SSH-enriched sequences. We found that approximately 9% of the WH8103 sequences were potential false-positive sequences, which demonstrated that caution should be used when this technology is used to assess genomic differences in genetically similar bacterial strains.