Polyphosphate- and polyhydroxyalkanoate (PHA)-accumulating traits of predominant microorganisms in an efficient enhanced biological phosphorus removal (EBPR) process were investigated systematically using a suite of non-culture-dependent methods. Results of 16S rDNA clone library and fluorescence in situ hybridization (FISH) with rRNA-targeted, group-specific oligonucleotide probes indicated that the microbial community consisted mostly of the alpha- (9.5% of total cells), beta- (41.3%) and gamma- (6.8%) subclasses of the class Proteobacteria, Flexibacter-Cytophaga (4.5%) and the Gram-positive high G+C (HGC) group (17.9%). With individual phylogenetic groups or subgroups, members of Candidatus Accumulibacter phosphatis in the beta-2 subclass, a novel HGC group closely related to Tetrasphaera spp., and a novel gamma-proteobacterial group were the predominant populations. Furthermore, electron microscopy with energy-dispersive X-ray analysis was used to validate the staining specificity of 4,6-diamino-2-phenylindole (DAPI) for intracellular polyphosphate and revealed the composition of polyphosphate granules accumulated in predominant bacteria as mostly P, Ca and Na. As a result, DAPI and PHA staining procedures could be combined with FISH to identify directly the polyphosphate- and PHA-accumulating traits of different phylogenetic groups. Members of Accumulibacter phosphatis and the novel gamma-proteobacterial group were observed to accumulate both polyphosphate and PHA. In addition, one novel rod-shaped group, closely related to coccus-shaped Tetrasphaera, and one filamentous group resembling Candidatus Nostocoidia limicola in the HGC group were found to accumulate polyphosphate but not PHA. No cellular inclusions were detected in most members of the alpha-Proteobacteria and the Cytophaga-Flavobacterium group. The diversified functional traits observed suggested that different substrate metabolisms were used by predominant phylogenetic groups in EBPR processes.
Acute hepatopancreatic necrosis disease (AHPND) (formerly, early mortality syndrome) is a high-mortality-rate shrimp disease prevalent in shrimp farming areas. Although AHPND is known to be caused by pathogenic Vibrio parahaemolyticus hosting the plasmid-related PirABvp toxin gene, the effects of disturbances in microbiome have not yet been studied. We took 62 samples from a grow-out pond during an AHPND developing period from Days 23 to 37 after stocking white postlarvae shrimp and sequenced the 16S rRNA genes with Illumina sequencing technology. The microbiomes of pond seawater and shrimp stomachs underwent varied dynamic succession during the period. Despite copies of PirABvp, principal co-ordinates analysis revealed two distinctive stages of change in stomach microbiomes associated with AHPND. AHPND markedly changed the bacterial diversity in the stomachs; it decreased the Shannon index by 53.6% within approximately 7 days, shifted the microbiome with Vibrio and Candidatus Bacilloplasma as predominant populations, and altered the species-to-species connectivity and complexity of the interaction network. The AHPND-causing Vibrio species were predicted to develop a co-occurrence pattern with several resident and transit members within Candidatus Bacilloplasma and Cyanobacteria. This study’s insights into microbiome dynamics during AHPND infection can be valuable for minimising this disease in shrimp farming ponds.
White spot syndrome virus (WSSV) is a virulent pathogen known to infect various crustaceans. It has bacilliform morphology with a tail-like appendage at one end. The envelope consists of four major proteins. Envelope structural proteins play a crucial role in viral infection and are believed to be the first molecules to interact with the host. Here, we report the localization and crystal structure of major envelope proteins VP26 and VP28 from WSSV at resolutions of 2.2 and 2.0 Å, respectively. These two proteins alone account for approximately 60% of the envelope, and their structures represent the first two structural envelope proteins of WSSV. Structural comparisons among VP26, VP28, and other viral proteins reveal an evolutionary relationship between WSSV envelope proteins and structural proteins from other viruses. Both proteins adopt -barrel architecture with a protruding N-terminal region. We have investigated the localization of VP26 and VP28 using immunoelectron microscopy. This study suggests that VP26 and VP28 are located on the outer surface of the virus and are observed as a surface protrusion in the WSSV envelope, and this is the first convincing observation for VP26. Based on our studies combined with the literature, we speculate that the predicted N-terminal transmembrane region of VP26 and VP28 may anchor on the viral envelope membrane, making the core -barrel protrude outside the envelope, possibly to interact with the host receptor or to fuse with the host cell membrane for effective transfer of the viral infection. Furthermore, it is tempting to extend this host interaction mode to other structural viral proteins of similar structures. Our finding has the potential to extend further toward drug and vaccine development against WSSV.In the shrimp aquaculture industry, more than 20 viruses have been reported to date. Of these, white spot syndrome virus (WSSV) is the major and most serious pathogen (4,5,24). The virulence of WSSV is very high, resulting in mortality rates from 90 to 100% within 3 to 7 days of infection and leading to complete devastation of the shrimp culture industry. This virus has a wide range of hosts among crustaceans and induces distinctive clinical signs of white spots on the carapace, appendages, and the inner surface of the body. WSSV belongs to a new virus family, Nimaviridae, under a new genus Whispovirus (www.ncbi.nih.gov/ICTVdb/Ictv/index.htm), which shares a low homology with other known DNA viruses (25, 41). WSSV is an enveloped virus with a 305-kb double-stranded circular DNA genome (41,46) and approximately 180 open reading frames. However, most of its presumptive open reading frames do not have any homology with known proteins and are functionally unknown.In order to understand the mechanism of WSSV infection and morphogenesis, we focused on the characterization of structural proteins. Among these structural proteins, the envelope proteins are extremely important because they are believed to be the first molecules to interact with the host and, consequently, pl...
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