Flavobacteria (members of the family Flavobacteriaceae ) dominate the bacterial community in the Anopheles mosquito midgut. One such commensal, Elizabethkingia anophelis , is closely associated with Anopheles mosquitoes through transstadial persistence (i.e., from one life stage to the next); these and other properties favor its development for paratransgenic applications in control of malaria parasite transmission. However, the physiological requirements of E. anophelis have not been investigated, nor has its capacity to perpetuate despite digestion pressure in the gut been quantified. To this end, we first developed techniques for genetic manipulation of E. anophelis , including selectable markers, reporter systems (green fluorescent protein [GFP] and NanoLuc), and transposons that function in E. anophelis . A flavobacterial expression system based on the promoter P ompA was integrated into the E. anophelis chromosome and showed strong promoter activity to drive GFP and NanoLuc reporter production. Introduced, GFP-tagged E. anophelis associated with mosquitoes at successive developmental stages and propagated in Anopheles gambiae and Anopheles stephensi but not in Aedes triseriatus mosquitoes. Feeding NanoLuc-tagged cells to A. gambiae and A. stephensi in the larval stage led to infection rates of 71% and 82%, respectively. In contrast, a very low infection rate (3%) was detected in Aedes triseriatus mosquitoes under the same conditions. Of the initial E. anophelis cells provided to larvae, 23%, 71%, and 85% were digested in A. stephensi , A. gambiae , and Aedes triseriatus , respectively, demonstrating that E. anophelis adapted to various mosquito midgut environments differently. Bacterial cell growth increased up to 3-fold when arginine was supplemented in the defined medium. Furthermore, the number of NanoLuc-tagged cells in A. stephensi significantly increased when arginine was added to a sugar diet, showing it to be an important amino acid for E. anophelis . Animal erythrocytes promoted E. anophelis growth in vivo and in vitro , indicating that this bacterium could obtain nutrients by participating in erythrocyte lysis in the mosquito midgut.

Dietary zinc oxide (ZnO) at pharmacological level has been widely used to prevent and treat diarrhea in weaning piglets. Despite its importance for promoting animal health and performance, the influence of microbiome profiles in intestinal tracts by ZnO needs to be comprehensively investigated. In this study, we conducted a comparative microbial community analysis in the ileum and colon of piglets fed by either control diet, high ZnO (3,000 mg/kg) supplement or antibiotics (300 mg/kg chlortetracycline and 60 mg/kg colistin sulfate) supplement. Our results showed that both high dietary ZnO and in-feed antibiotics supplementations significantly increased 5 phyla of Spirochaetes, Tenericutes, Euryarchaeota, Verrucomicrobia, TM7 and reduced 1 phyla of Chlamydiae in ileal digesta. The relative abundance of opportunistic pathogens Campylobacterales were decreased while Enterobacteriales were increased in ZnO or antibiotics-supplemented group when compared to the control. In the colon, the phyla Euryarchaeota, the genus Methanobrevibacter, and the species Methanobrevibacter smithii were drastically increased by high dietary ZnO supplementation when compared with other groups. The microbial functional prediction analysis showed that high dietary ZnO and in-feed antibiotics had a higher abundance of transporter pathway enrichment in the ileum when compared with the control. While in the colon high dietary ZnO had a higher abundant enrichment of methane metabolism involving energy supply when compared with other groups. Both high dietary ZnO and antibiotics increased the microbiota diversity of ileal digesta while they decreased the microbiota diversity of the colonic digesta. Collectively, these results suggested that dietary ZnO and in-feed antibiotics supplementations presented similar effect on ileal microbiota, and mainly affected the non-predominant microbiota.

Strains of Serratia marcescens, originally isolated from the gut lumen of adult female Anopheles stephensi mosquitoes, established persistent infection at high rates in adult A. stephensi whether fed to larvae or in the sugar meal to adults. By contrast, the congener S. fonticola originating from Aedes triseriatus had lower infection in A. stephensi, suggesting co-adaptation of Serratia strains in different species of host mosquitoes. Coinfection at high infection rate in adult A. stephensi resulted after feeding S. marcescens and Elizabethkingia anophelis in the sugar meal, but when fed together to larvae, infection rates with E. anophelis were much higher than were S. marcescens in adult A. stephensi, suggesting a suppression effect of coinfection across life stages. A primary isolate of S. marcescens was resistant to all tested antibiotics, showed high survival in the mosquito gut, and produced alpha-hemolysins which contributed to lysis of erythrocytes ingested with the blood meal. Genomes of two primary isolates from A. stephensi, designated S. marcescens ano1 and ano2, were sequenced and compared to other Serratia symbionts associated with insects, nematodes and plants. S. marcescens ano1 and ano2 had predicted virulence factors possibly involved in attacking parasites and/or causing opportunistic infection in mosquito hosts. S. marcescens ano1 and ano2 possessed multiple mechanisms for antagonism against other microorganisms, including production of bacteriocins and multi-antibiotic resistance determinants. These genes contributing to potential anti-malaria activity including serralysins, hemolysins and chitinases are only found in some Serratia species. It is interesting that genome sequences in S. marcescens ano1 and ano2 are distinctly different from those in Serratia sp. Ag1 and Ag2 which were isolated from Anopheles gambiae. Compared to Serratia sp. Ag1 and Ag2, S. marcescens ano1 and ano2 have more rRNAs and many important genes involved in commensal and anti-parasite traits.

Bacterial outer membrane vesicles (OMV) are packets of periplasmic material that, via the proteins and other molecules they contain, project metabolic function into the environment. While OMV production is widespread in proteobacteria, they have been extensively studied only in pathogens, which inhabit fully hydrated environments. However, many (arguably most) bacterial habitats, such as soil, are only partially hydrated. In the latter, water is characteristically distributed as films on soil particles that are, on average thinner, than are typical OMV (ca. ≤10 nm water film vs. 20 to >200 nm OMV;). We have identified a new bacterial surface structure, termed a “nanopod”, that is a conduit for projecting OMV significant distances (e.g., ≥6 µm) from the cell. Electron cryotomography was used to determine nanopod three-dimensional structure, which revealed chains of vesicles within an undulating, tubular element. By using immunoelectron microscopy, proteomics, heterologous expression and mutagenesis, the tubes were determined to be an assembly of a surface layer protein (NpdA), and the interior structures identified as OMV. Specific metabolic function(s) for nanopods produced by Delftia sp. Cs1-4 are not yet known. However, a connection with phenanthrene degradation is a possibility since nanopod formation was induced by growth on phenanthrene. Orthologs of NpdA were identified in three other genera of the Comamonadaceae family, and all were experimentally verified to form nanopods. Nanopods are new bacterial organelles, and establish a new paradigm in the mechanisms by which bacteria effect long-distance interactions with their environment. Specifically, they create a pathway through which cells can effectively deploy OMV, and the biological activity these transmit, in a diffusion-independent manner. Nanopods would thus allow environmental bacteria to expand their metabolic sphere of influence in a manner previously unknown for these organisms.

Cloning of a laccase-encoding cDNA from the edible straw mushroom, Volvariella volvacea, was performed using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends. The cDNA of the putative laccase gene (lac4) consisted of 1689 bp, including an open reading frame encoding a 23-amino acid signal peptide at the N-terminal end and a 540-amino acid mature protein with a predicted molecular mass of 58173 Da and a pI value of 6.1. The 10 histidine residues and one cysteine residue required to co-ordinate the four copper atoms at the active site of the protein were all conserved. The amino acid sequence of V. volvacea lac4 has a high degree of identity with other basidiomycete laccases. Transcription of the laccase gene was analysed by RT-PCR and, unlike many other laccase genes, shown to be regulated independently of either copper or aromatic compounds under the test conditions. However, the laccase gene is strongly expressed during that part of the mushroom developmental cycle involving fruit body morphogenesis.