Engineering Parageobacillus thermoglucosidans as a robust platform for bioethanol production

Philosophiae Doctor - PhD (Biotechnology)Parageobacillus thermoglucosidans is a promising “platform” organism to use in the production ofa range of useful metabolites with demonstrated ability to produce ethanol, isobutanol and polylactic acid for bio-degradable plastics. Extensive work has been done in engineering the organism for enhanced ethanol production. However, an often used and highly effective alternative pathway (pyruvate decarboxylase mediated) for ethanol production has not yet been demonstrated in P. thermoglucosidans. We first characterize two novel bacterial pyruvate decarboxylase enzymes (PDC’s) then attempt to express the more thermostable of these enzymes from Gluconobacter oxydans in P. thermoglucosidans to improve ethanol yields. Initial expression was unsuccessful. Analysis of the codon usage pattern for the gene revealed that the codon usage was suboptimal in the heterologous host P. thermoglucosidans. After codon harmonization, we could demonstrate successful expression of the enzyme at 45°C, however not at the bacterium’s optimum growth temperature of 60°C. This was concomitant with enhanced ethanol production close to the theoretical yield possible (0.5g/l)

Microbiome enrichment from contaminated marine sediments unveils novel bacterial strains for petroleum hydrocarbon and heavy metal bioremediation

Petroleum hydrocarbons and heavy metals are some of the most widespread contaminants affecting marine ecosystems, urgently needing effective and sustainable remediation solutions. Microbial-based bioremediation is gaining increasing interest as an effective, economically and environmentally sustainable strategy. Here, we hypothesized that the heavily polluted coastal area facing the Sarno River mouth, which discharges >3 tons of polycyclic aromatic hydrocarbons (PAHs) and ~15 tons of heavy metals (HMs) into the sea annually, hosts unique microbiomes including marine bacteria useful for PAHs and HMs bioremediation. We thus enriched the microbiome of marine sediments, contextually selecting for HM-resistant bacteria. The enriched mixed bacterial culture was subjected to whole-DNA sequencing, metagenome-assembled-genomes (MAGs) annotation, and further sub-culturing to obtain the major bacterial species as pure strains. We obtained two novel isolates corresponding to the two most abundant MAGs (Alcanivorax xenomutans strain-SRM1 and Halomonas alkaliantarctica strain-SRM2), and tested their ability to degrade PAHs and remove HMs. Both strains exhibited high PAHs degradation (60–100%) and HMs removal (21–100%) yield, and we described in detail >60 genes in their MAGs to unveil the possible genetic basis for such abilities

Degradation of hydrocarbons and heavy metal reduction by marine bacteria in highly contaminated sediments

Investigations on the ability of bacteria to enhance removal of hydrocarbons and reduce heavy metal toxicity in sediments are necessary to design more effective bioremediation strategies. In this study, five bacterial strains, Halomonas sp. SZN1, Alcanivorax sp. SZN2, Pseudoalteromonas sp. SZN3, Epibacterium sp. SZN4, and Virgibacillus sp. SZN7, were isolated from polluted sediments from an abandoned industrial site in the Gulf of Naples, Mediterranean Sea, and tested for their bioremediation efficiency on sediment samples collected from the same site. These bacteria were added as consortia or as individual cultures into polluted sediments to assess biodegradation efficiency of polycyclic aromatic hydrocarbons and heavy metal immobilisation capacity. Our results indicate that these bacteria were able to remove polycyclic aromatic hydrocarbons, with a removal rate up to ca. 80% for dibenzo-anthracene

Genomic characterization of a prophage, Smhb1, that Infects Salinivibrio kushneri BNH isolated from a Namib Desert saline spring

Funding text 1 Funding: This work was supported by the National Research Foundation (NRF) of South Africa. Funding text 2 This work was supported by the National Research Foundation (NRF) of South Africa. We wish to thank the researcher and management of the Gobabeb Desert research station for hosting us during sample collection. We also thank Mohammed Jaffer at the Electron Microscope Unit at the University of Cape Town for helping to capture TEM images.Recent years have seen the classification and reclassification of many viruses related to the model enterobacterial phage P2. Here, we report the identification of a prophage (Smhb1) that infects Salinivibrio kushneri BNH isolated from a Namib Desert salt pan (playa). Analysis of the genome revealed that it showed the greatest similarity to P2-like phages that infect Vibrio species and showed no relation to any of the previously described Salinivibrio-infecting phages. Despite being distantly related to these Vibrio infecting phages and sharing the same modular gene arrangement as seen in most P2-like viruses, the nucleotide identity to its closest relatives suggest that, for now, Smhb1 is the lone member of the Peduovirus genus Playavirus. Although host range testing was not extensive and no secondary host could be identified for Smhb1, genomic evidence suggests that the phage is capable of infecting other Salinivibrio species, including Salinivibrio proteolyticus DV isolated from the same playa. Taken together, the analysis presented here demonstrates how adaptable the P2 phage model can be. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.National Research Foundation (NRF University of Cape Town (UCT

Isolation and characterization of strain exiguobacterium sp. Krl4, a producer of bioactive secondary metabolites from a Tibetan glacier

Extremophilic microorganisms represent a unique source of novel natural products. Among them, cold adapted bacteria and particularly alpine microorganisms are still underexplored. Here, we describe the isolation and characterization of a novel Gram-positive, aerobic rod-shaped alpine bacterium (KRL4), isolated from sediments from the Karuola glacier in Tibet, China. Complete phenotypic analysis was performed revealing the great adaptability of the strain to a wide range of temperatures (5–40 ◦C), pHs (5.5–8.5), and salinities (0–15% w/v NaCl). Genome sequencing identified KRL4 as a member of the placeholder genus Exiguobacterium_A and annotation revealed that only half of the protein-encoding genes (1522 of 3079) could be assigned a putative function. An analysis of the secondary metabolite clusters revealed the presence of two uncharacterized phytoene synthase containing pathways and a novel siderophore pathway. Biological assays confirmed that the strain produces molecules with antioxidant and siderophore activities. Furthermore, intracellular extracts showed nematocidal activity towards C. elegans, suggesting that strain KRL4 is a source of anthelmintic compounds

Three novel bacteria associated with two centric diatom species from the Mediterranean Sea, Thalassiosira rotula and Skeletonema marinoi

Diatoms are a successful group of microalgae at the base of the marine food web. For hundreds of millions of years, they have shared common habitats with bacteria, which favored the onset of interactions at different levels, potentially driving the synthesis of biologically active molecules. To unveil their presence, we sequenced the genomes of bacteria associated with the centric diatom Thalassiosira rotula from the Gulf of Naples. Annotation of the metagenome and its analysis allowed the reconstruction of three bacterial genomes that belong to currently undescribed species. Their investigation showed the existence of novel gene clusters coding for new polyketide molecules, antibiotics, antibiotic-resistance genes and an ectoine production pathway. Real-time PCR was used to investigate the association of these bacteria with three different diatom clones and revealed their preference for T. rotula FE80 and Skeletonema marinoi FE7, but not S. marinoi FE60 from the North Adriatic Sea. Additionally, we demonstrate that although all three bacteria could be detected in the culture supernatant (free-living), their number is up to 45 times higher in the cell associated fraction, suggesting a close association between these bacteria and their host

Antibacterial activities of bacteria isolated from the marine sponges Isodictya compressa and Higginsia bidentifera collected from Algoa Bay, South Africa

Due to the rise inmulti-drug resistant pathogens and other diseases, there is renewed interest in marine sponge endosymbionts as a rich source of natural products (NPs). The South African marine environment is rich in marine biota that remains largely unexplored and may represent an important source for the discovery of novel NPs.We first investigated the bacterial diversity associated with five South African marine sponges, whose microbial populations had not previously been investigated, and select the two sponges (Isodictya compressa and Higginsia bidentifera) with highest species richness to culture bacteria. By employing 33 different growth conditions 415 sponge-associated bacterial isolates were cultured and screened for antibacterial activity. Thirty-five isolates showed antibacterial activity, twelve of which exhibited activity against the multi-drug resistant Escherichia coli 1699, implying that some of the bioactive compounds could be novel. Genome sequencing of two of these isolates confirmed that they harbour uncharacterized biosynthetic pathways that may encode novel chemical structures.IS

Degradation of Hydrocarbons and Heavy Metal Reduction by Marine Bacteria in Highly Contaminated Sediments

Investigations on the ability of bacteria to enhance removal of hydrocarbons and reduce heavy metal toxicity in sediments are necessary to design more effective bioremediation strategies. In this study, five bacterial strains, Halomonas sp. SZN1, Alcanivorax sp. SZN2, Pseudoalteromonas sp. SZN3, Epibacterium sp. SZN4, and Virgibacillus sp. SZN7, were isolated from polluted sediments from an abandoned industrial site in the Gulf of Naples, Mediterranean Sea, and tested for their bioremediation efficiency on sediment samples collected from the same site. These bacteria were added as consortia or as individual cultures into polluted sediments to assess biodegradation efficiency of polycyclic aromatic hydrocarbons and heavy metal immobilisation capacity. Our results indicate that these bacteria were able to remove polycyclic aromatic hydrocarbons, with a removal rate up to ca. 80% for dibenzo-anthracene. In addition, these bacteria reduced arsenic, lead, and cadmium mobility by promoting their partitioning into less mobile and bioavailable fractions. Microbial consortia generally showed higher performance toward pollutants as compared with pure isolates, suggesting potential synergistic interactions able to enhance bioremediation capacity. Overall, our findings suggest that highly polluted sediments select for bacteria efficient at reducing the toxicity of hazardous compounds, paving the way for scaled-up bioremediation trials

Three novel bacteriophages isolated from the East African Rift Valley soda lakes

BACKGROUND : Soda lakes are unique environments in terms of their physical characteristics and the biology they harbour. Although well studied with respect to their microbial composition, their viral compositions have not, and consequently few bacteriophages that infect bacteria from haloalkaline environments have been described. METHODS : Bacteria were isolated from sediment samples of lakes Magadi and Shala. Three phages were isolated on two different Bacillus species and one Paracoccus species using agar overlays. The growth characteristics of each phage in its host was investigated and the genome sequences determined and analysed by comparison with known phages. RESULTS : Phage Shbh1 belongs to the family Myoviridae while Mgbh1 and Shpa belong to the Siphoviridae family. Tetranucleotide usage frequencies and G + C content suggests that Shbh1 and Mgbh1 do not regularly infect, and have therefore not evolved with, the hosts they were isolated on here. Shbh1 was shown capable of infecting two different Bacillus species from the two different lakes demonstrating its potential broad-host range. Comparative analysis of their genome sequence with known phages revealed that, although novel, Shbh1 does share substantial amino acid similarity with previously described Bacillus infecting phages (Grass, phiNIT1 and phiAGATE) and belongs to the Bastille group, while Mgbh1 and Shpa are highly novel. CONCLUSION : The addition of these phages to current databases should help with metagenome/metavirome annotation efforts. We describe a highly novel Paracoccus infecting virus (Shpa) which together with NgoΦ6 and vB_PmaS_IMEP1 is one of only three phages known to infect Paracoccus species but does not show similarity to these phages.Additional file 1: Figure S1. Whole genome alignment of phage Shbh1 with six of its closest relatives. Similarly coloured regions indicate homology or local collinear blocks (LCB) between nucleotide sequences, with the level of similarity indicated by the height of the bars within each LCB. Genome alignments were performed using MAUVE.Additional file 2: Table S1. Predicted open reading frames on Shpa and closest BLASTp hit on the NCBI database.Additional file 3: Table S2. Predicted open reading frames on Mgbh1 and closest BLASTp hit on the NCBI database.Additional file 4: Table S3. Predicted open reading frames on Shbh1 and closest BLASTp hit on the NCBI database.Additional file 5: Figure S2. GC skew analysis of the Shbh1 genome showing putative replication origin (ori) and termination sites (ter) calculated using a window size of 1000 bp and a step size of 100 bp.Additional file 6: Figure S4. GC skew analysis of the Shpa genome showing putative replication origin (ori) and termination sites (ter) calculated using a window size of 1000 bp and a step size of 100 bp.Additional file 7: Figure S3. GC skew analysis of the Mgbh1 genome showing putative replication origin (ori) and termination sites (ter) calculated using a window size of 1000 bp and a step size of 100 bp.The National Research Foundation (NRF) of South Africahttp://www.virologyj.comam2017Genetic