Stable Bacterial Cultures for Producing Alginates

Methods for mass producing bacterial alginate, bacterial cultures for producing alginate, and pharmaceutical compositions containing bacterial alginate are contemplated

RpoN (σ54) Is Required for Floc Formation but Not for Extracellular Polysaccharide Biosynthesis in a Floc-Forming Aquincola tertiaricarbonis Strain.

Some bacteria are capable of forming flocs, in which bacterial cells become self-flocculated by secreted extracellular polysaccharides and other biopolymers. The floc-forming bacteria play a central role in activated sludge, which has been widely utilized for the treatment of municipal sewage and industrial wastewater. Here, we use a floc-forming bacterium, Aquincolatertiaricarbonis RN12, as a model to explore the biosynthesis of extracellular polysaccharides and the regulation of floc formation. A large gene cluster for exopolysaccharide biosynthesis and a gene encoding the alternative sigma factor RpoN1, one of the four paralogues, have been identified in floc formation-deficient mutants generated by transposon mutagenesis, and the gene functions have been further confirmed by genetic complementation analyses. Interestingly, the biosynthesis of exopolysaccharides remained in the rpoN1-disrupted flocculation-defective mutants, but most of the exopolysaccharides were secreted and released rather than bound to the cells. Furthermore, the expression of exopolysaccharide biosynthesis genes seemed not to be regulated by RpoN1. Taken together, our results indicate that RpoN1 may play a role in regulating the expression of a certain gene(s) involved in the self-flocculation of bacterial cells but not in the biosynthesis and secretion of exopolysaccharides required for floc formation.IMPORTANCE Floc formation confers bacterial resistance to predation of protozoa and plays a central role in the widely used activated sludge process. In this study, we not only identified a large gene cluster for biosynthesis of extracellular polysaccharides but also identified four rpoN paralogues, one of which (rpoN1) is required for floc formation in A. tertiaricarbonis RN12. In addition, this RpoN sigma factor regulates the transcription of genes involved in biofilm formation and swarming motility, as previously shown in other bacteria. However, this RpoN paralogue is not required for the biosynthesis of exopolysaccharides, which are released and dissolved into culture broth by the rpoN1 mutant rather than remaining tightly bound to cells, as observed during the flocculation of the wild-type strain. These results indicate that floc formation is a regulated complex process, and other yet-to-be identified RpoN1-dependent factors are involved in self-flocculation of bacterial cells via exopolysaccharides and/or other biopolymers

The Pseudomonas aeruginosa Sensor Kinase KinB Negatively Controls Alginate Production through AlgW-Dependent MucA Proteolysis▿

Mucoidy, or overproduction of the exopolysaccharide known as alginate, in Pseudomonas aeruginosa is a poor prognosticator for lung infections in cystic fibrosis. Mutation of the anti-σ factor MucA is a well-accepted mechanism for mucoid conversion. However, certain clinical mucoid strains of P. aeruginosa have a wild-type (wt) mucA. Here, we describe a loss-of-function mutation in kinB that causes overproduction of alginate in the wt mucA strain PAO1. KinB is the cognate histidine kinase for the transcriptional activator AlgB. Increased alginate production due to inactivation of kinB was correlated with high expression at the alginate-related promoters PalgU and PalgD. Deletion of alternative σ factor RpoN (σ54) or the response regulator AlgB in kinB mutants decreased alginate production to wt nonmucoid levels. Mucoidy was restored in the kinB algB double mutant by expression of wt AlgB or phosphorylation-defective AlgB.D59N, indicating that phosphorylation of AlgB was not required for alginate overproduction when kinB was inactivated. The inactivation of the DegS-like protease AlgW in the kinB mutant caused loss of alginate production and an accumulation of the hemagglutinin (HA)-tagged MucA. Furthermore, we observed that the kinB mutation increased the rate of HA-MucA degradation. Our results also indicate that AlgW-mediated MucA degradation required algB and rpoN in the kinB mutant. Collectively, these studies indicate that KinB is a negative regulator of alginate production in wt mucA strain PAO1

PBAD-Based Shuttle Vectors for Functional Analysis of Toxic and Highly Regulated Genes in Pseudomonas and Burkholderia spp. and Other Bacteria▿

We report the construction of a series of Escherichia-Pseudomonas broad-host-range expression vectors utilizing the PBAD promoter and the araC regulator for routine cloning, conditional expression, and analysis of tightly controlled and/or toxic genes in pseudomonads

Disodium 2-oxoglutarate promotes carbon flux into astaxanthin and fatty acid biosynthesis pathways in Haematococcus

Improving carbon availability in astaxanthin production is pivotal in Haematococcus industry. In this study, disodium 2-oxoglutarate (2-OG-2Na) was observed to be a potential carbon regulator to increase the astaxanthin content. To illustrate its efficacy in astaxanthin production, key genes and enzyme were analyzed. Upon 2-OG-2Na treatment, genes ipi, bkt and crtR-b were up regulated, concomitantly, carotenoids and astaxanthin content increased by 15.4% and 14.0% at 120 h, respectively; additionally, Acetyl-CoA carboxylase was activated, consistent with 1.27-fold increase in fatty acids content. PUFAs increased earlier as fatty acids assembly gene fad was up-regulated to 20.56. It was also found that cell division was not compromised. Altogether, it was suggested that increased carbon skeletons were re-directed into the astaxanthin and fatty acids biosynthesis pathway. Furthermore, 2-OG-2Na was applied in ten Haematococcus strains. Of these strains, astaxanthin contents were accelerated with average net increase of 10.48%, exhibiting a scalable paradigm for commercial production.</p

Effects of salinity on the performance of bioflocs with activated sludge as inoculum

This study evaluated the feasibility of domesticating the bioflocs with activated sludge as inoculum at high salinity. By using the gradually increase salinity from 0.2 to 4.0%, the study evaluated the effectiveness of bioflocs in sustaining the water quality of aquaculture, the biofloc morphology characteristics and microbial community structure of bioflocs in order to discover the influence of salinity. From the perspective of sustaining the water quality of bioflocs, the COD removal efficiency dropped sharply from 63.9% to 3.7%, the NO3−-N was maintained below 0.5 mg/L but the NH4+-N exceeded the safety threshold of aquaculture at the salinity of 2.5–4.0%. From the pespective of flocculation of flocs, the biofloc volume index and content were maintained at about 30 ml/g and 7 g/L, while the floc particle size (45–200 um) tended to increase cumulatively, showing good agglomeration, sedimentation and stability. From the pespective of floc microbial community structure, Arenibacter, Thauera, Paracoccus and Denitromonas became the dominant genera with relative abundances of 4.8–7.5%, 4.9–17.1%, 3.0–4.7% and 5.3–14.1% at 3.0–4.0% salinity, respectively, however, the relative abundance of Candidatus Competibacter rapidly decreased from 15.0% to 2.5% with the increasing salinity from 1.0% to 4.0%. Furthermore, Redundancy analysis (RDA) indicated that salinity was a key environmental factor affecting floc community, and Functional Annotation of Prokaryotic Taxa (FAPROTAX) confirmed the promoted flocs denitrification as well as inhibited nitrification and hydrocarbon cycling in higher salinity to some extent. This study demonstrated the feasibility of using freshwater activated sludge as a base nucleus for biofloc formation for salinity up to 2% – 2.5%, which provided a useful reference for improving the taste and nutritional value of fish cultured by Biofloc Technology (BFT)

An extracytoplasmic function sigma factor-dependent periplasmic glutathione peroxidase is involved in oxidative stress response of Shewanella oneidensis

AbstractBackgroundBacteria use alternative sigma factors (σs) to regulate condition-specific gene expression for survival and Shewanella harbors multiple ECF (extracytoplasmic function) σ genes and cognate anti-sigma factor genes. Here we comparatively analyzed two of the rpoE-like operons in the strain MR-1: rpoE-rseA-rseB-rseC and rpoE2-chrR.ResultsRpoE was important for bacterial growth at low and high temperatures, in the minimal medium, and high salinity. The degP/htrA orthologue, required for growth of Escherichia coli and Pseudomonas aeruginosa at high temperature, is absent in Shewanella, while the degQ gene is RpoE-regulated and is required for bacterial growth at high temperature. RpoE2 was essential for the optimal growth in oxidative stress conditions because the rpoE2 mutant was sensitive to hydrogen peroxide and paraquat. The operon encoding a ferrochelatase paralogue (HemH2) and a periplasmic glutathione peroxidase (PgpD) was identified as RpoE2-dependent. PgpD exhibited higher activities and played a more important role in the oxidative stress responses than the cytoplasmic glutathione peroxidase CgpD under tested conditions. The rpoE2-chrR operon and the identified regulon genes, including pgpD and hemH2, are coincidently absent in several psychrophilic and/or deep-sea Shewanella strains.ConclusionIn S. oneidensis MR-1, the RpoE-dependent degQ gene is required for optimal growth under high temperature. The rpoE2 and RpoE2-dependent pgpD gene encoding a periplasmic glutathione peroxidase are involved in oxidative stress responses. But rpoE2 is not required for bacterial growth at low temperature and it even affected bacterial growth under salt stress, indicating that there is a tradeoff between the salt resistance and RpoE2-mediated oxidative stress responses

An RpoN-dependent PEP-CTERM gene is involved in floc formation of an Aquincola tertiaricarbonis strain

Abstract Background The floc is a characteristic of microbial aggregate growth, displaying cloudy suspensions in water. Floc formation has been demonstrated in a series of bacteria and the floc-forming bacteria play a crucial role in activated sludge (AS) process widely used for municipal sewage and industrial wastewater treatment over a century. It has been demonstrated that some exopolysaccharide biosynthesis genes and the sigma factor (sigma54 or rpoN) were required for floc forming in some bacteria. However, the mechanism underlying the floc formation stills need to be elucidated. Results In this study, we demonstrate that a TPR (Tetratricopeptide repeats) protein-encoding gene prsT is required for floc formation of Aquincola tertiaricarbonis RN12 and an upstream PEP-CTERM gene (designated pepA), regulated by RpoN1, is involved in its floc formation but not swarming motility and biofilm formation. Overexpression of PepA could rescue the floc-forming phenotype of the rpoN1 mutant by decreasing the released soluble exopolysaccharides and increasing the bound polymers. Conclusion Our results indicate that the wide-spread PEP-CTERM proteins play an important role in the self-flocculation of bacterial cells and may be a component of extracellular polymeric substances required for floc-formation