Functional traits of particle-associated microbial assemblages in the low-latitude Pacific Ocean

Background Organic particles are hotspots for microbial activity and serve as sites of organic matter mineralisation in the water column of marine systems. In nutrient-limited surface water, degradation of organic matter and nutrient regeneration by marine microbes is crucial. Although free-living (FL) bacteria vastly outnumber those on particles, particle-associated (PA) bacteria can reach locally higher concentrations. Accordingly, to achieve a better understanding of marine microbial ecosystems, it is important to elucidate the differences in not only microbial community structures, but also functional traits, between PA and FL environmental sample fractions. In a previous study, we demonstrated that the Genomaple (formerly MAPLE) system could successfully differentiate the functional potentials and diversity of contributors to each function in four metagenomic datasets generated by the Global Ocean Sampling expedition. Hence, we also used this system to highlight functional traits in PA microbial assemblages. Results The PA and FL fractions could be distinguished from one another by their taxonomic compositions, inferred from ribosomal proteins and relative abundance of module functions. Module functions that were more abundant among PA assemblages than FL assemblages were shared between both subtropical gyres, and their taxonomic compositions were similar. Bacterial transport systems associated with adhesive molecules used for forming microbial assemblages through particulate organic matter were more abundant in the PA fractions. Bacterial regulatory system elements for C 4 -dicarboxylate transport and B-vitamin biosynthesis were also abundant among PA assemblages, suggesting mutual relationships between bacteria and algae involved in exchange of nutrient sources. On the other hand, module functions related to amino acid biosynthesis and bacterial transport systems for inorganic nitrogen, phosphorus, and urea were significantly more abundant in the PA assemblages of more oligotrophic North and South Pacific subtropical gyres than eastern equatorial Pacific regions. Conclusions Comprehensive functional metagenomic analyses based on functional abundance revealed some notable functional traits in PA assemblages related to cell adhesion and nutrient acquisition, enabling the microbes to survive in subtropical regions that are more oligotrophic than the equatorial regions.

Dicarboxylate Transporters of Azospirillum brasilense Sp7 Play an Important Role in the Colonization of Finger Millet (Eleusine coracana) Roots

Azospirillum brasilense is a plant growth–promoting bacterium that colonizes the roots of a large number of plants, including C3 and C4 grasses. Malate has been used as a preferred source of carbon for the enrichment and isolation Azospirillum spp., but the genes involved in their transport and utilization are not yet characterized. In this study, we investigated the role of the two types of dicarboxylate transporters (DctP and DctA) of A. brasilense in their ability to colonize and promote growth of the roots of a C4 grass. We found that DctP protein was distinctly upregulated in A. brasilense grown with malate as sole carbon source. Inactivation of dctP in A. brasilense led to a drastic reduction in its ability to grow on dicarboxylates and form cell aggregates. Inactivation of dctA, however, showed a marginal reduction in growth and flocculation. The growth and nitrogen fixation of a dctP and dctA double mutant of A. brasilense were severely compromised. We have shown here that DctPQM and DctA transporters play a major and a minor role in the transport of C4-dicarboxylates in A. brasilense, respectively. Studies on inoculation of the seedlings of a C4 grass, Eleusine corcana, with A. brasilense and its dicarboxylate transport mutants revealed that dicarboxylate transporters are required by A. brasilense for an efficient colonization of plant roots and their growth.

Bradyrhizobium diazoefficiens USDA 110-Glycine max interactome provides candidate proteins associated with symbiosis

Although the legume-rhizobium symbiosis is a most important biological process, there is a limited knowledge about the protein interaction network between host and symbiont. Using interolog and domain-based approaches, we constructed an inter-species protein interactome with 5115 protein-protein interactions between 2291 Glycine max and 290 Bradyrhizobium diazoefficiens USDA 110 proteins. The interactome was validated by expression pattern analysis in nodules, GO term semantic similarity, and co-expression analysis. One sub-network was further confirmed using luciferase complementation image assay. In the G. max-B. diazoefficiens interactome, bacterial proteins are mainly ion channel and transporters of carbohydrates and cations, while G. max proteins are mainly involved in the processes of metabolism, signal transduction, and transport. We also identified the top ten highly interacting proteins (hubs) for each of the two species. KEGG pathway analysis for each hub showed that two 14-3-3 proteins (SGF14g and SGF14k) and five heat shock proteins in G. max are possibly involved in symbiosis, and ten hubs in B. diazoefficiens may be important symbiotic effectors. Subnetwork analysis showed that 18 symbiosis-related SNARE proteins may play roles in regulating bacterial ion channels, and SGF14g and SGF14k possibly regulate the rhizobium dicarboxylate transport protein DctA. The predicted interactome and symbiosis proteins provide a valuable basis for understanding the molecular mechanism of root nodule symbiosis in soybean.

Expression of sodium-dependent dicarboxylate transporter 1 (NaDC1/SLC13A2) in normal and neoplastic human kidney

Regulated dicarboxylate transport is critical for acid-base homeostasis, prevention of calcium nephrolithiasis, regulation of collecting duct sodium chloride transport, and the regulation of blood pressure. Although luminal dicarboxylate reabsorption via NaDC1 (SLC13A2) is believed to be the primary mechanism regulating renal dicarboxylate transport, the specific localization of NaDC1 in the human kidney is currently unknown. This study’s purpose was to determine NaDC1's expression in normal and neoplastic human kidneys. Immunoblot analysis demonstrated NaDC1 expression with an apparent molecular weight of ~61 kDa. Immunohistochemistry showed apical NaDC1 immunolabel in the proximal tubule of normal human kidney tissue; well-preserved proximal tubule brush border was clearly labeled. Apical NaDC1 expression was evident throughout the entire proximal tubule, including the initial proximal convoluted tubule, as identified by origination from the glomerular tuft, and extending through the terminal of the proximal tubule, the proximal straight tubule in the outer medulla. We confirmed proximal tubule localization by colocalization with the proximal tubule specific protein, NBCe1. NaDC1 immunolabel was not detected other than in the proximal tubule. In addition, NaDC1 immunolabel was not detected in tumors of presumed proximal tubule origin, clear cell and papillary renal cell carcinoma, or in tumors of nonproximal tubule origin, oncocytoma and chromophobe carcinoma. In summary, 1) in the human kidney, apical NaDC1 immunolabel is present throughout the entire proximal tubule, and is not detectable in other renal cells; and 2) NaDC1 immunolabel is not present in renal tumors. These studies provide important information regarding NaDC1’s role in human dicarboxylate metabolism.

Detection of Goss’s Wilt Pathogen Clavibacter michiganensis subsp. nebraskensis in Maize by Loop-Mediated Amplification

The Goss’s wilt pathogen, Clavibacter michiganensis subsp. nebraskensis, can cause considerable losses in maize (Zea mays) production. Diagnosis of Goss’s wilt currently is based on symptomology and identification of C. michiganensis subsp. nebraskensis, following isolation on a semiselective medium and/or serological testing. In an effort to provide a more efficient identification method, a loop-mediated amplification (LAMP) assay was developed to detect the tripartite ATP-independent periplasmic (TRAP)-type C4-dicarboxylate transport system large permease component and tested using strains of C. michiganensis subsp. nebraskensis, all other C. michiganensis subspecies and several genera of nontarget bacteria. Only strains of C. michiganensis subsp. nebraskensis reacted positively with the LAMP assay. The LAMP assay was then used to identify bacterial isolates from diseased maize. 16S rDNA and dnaA sequence analyses were used to confirm the identity of the maize isolates and validate assay specificity. The Cmm ImmunoStrip assay was included as a presumptive identification test of C. michiganensis subsp. nebraskensis at the species level. The Cmn-LAMP assay was further tested using symptomatic leaf tissue. The Cmn-LAMP assay was run in a hand-held real-time monitoring device (SMART-DART) and performed equally to in-lab quantitative polymerase chain reaction equipment. The Cmn-LAMP assay accurately identified C. michiganensis subsp. nebraskensis and has potential as a field test. The targeted sequence also has potential application in other molecular detection platforms.

A Na+-coupled C4-dicarboxylate transporter (Asuc_0304) and aerobic growth of Actinobacillus succinogenes on C4-dicarboxylates

Actinobacillus succinogenes, which is known to produce large amounts of succinate during fermentation of hexoses, was able to grow on C4-dicarboxylates such as fumarate under aerobic and anaerobic conditions. Anaerobic growth on fumarate was stimulated by glycerol and the major product was succinate, indicating the involvement of fumarate respiration similar to succinate production from glucose. The aerobic growth on C4-dicarboxylates and the transport proteins involved were studied. Fumarate was oxidized to acetate. The genome of A. succinogenes encodes six proteins with similarity to secondary C4-dicarboxylate transporters, including transporters of the Dcu (C4-dicarboxylate uptake), DcuC (C4-dicarboxylate uptake C), DASS (divalent anion : sodium symporter) and TDT (tellurite resistance dicarboxylate transporter) family. From the cloned genes, Asuc_0304 of the DASS family protein was able to restore aerobic growth on C4-dicarboxylates in a C4-dicarboxylate-transport-negative Escherichia coli strain. The strain regained succinate or fumarate uptake, which was dependent on the electrochemical proton potential and the presence of Na+. The transport had an optimum pH ~7, indicating transport of the dianionic C4-dicarboxylates. Transport competition experiments suggested substrate specificity for fumarate and succinate. The transport characteristics for C4-dicarboxylate uptake by cells of aerobically grown A. succinogenes were similar to those of Asuc_0304 expressed in E. coli, suggesting that Asuc_0304 has an important role in aerobic fumarate uptake in A. succinogenes. Asuc_0304 has sequence similarity to bacterial Na+-dicarboxylate cotransporters and contains the carboxylate-binding signature. Asuc_0304 was named SdcA (sodium-coupled C4-dicarboxylate transporter from A . succinogenes).