Ingrid Bakke

PCR-based community structure studies of bacteria associated with eukaryotic organisms: a simple PCR strategy to avoid co-amplification of eukaryotic DNA.

PCR primers targeting conserved regions of the SSU rRNA gene are commonly used in bacterial community studies. For microbes associated with eukaryotes, co-amplification of eukaryotic DNA may preclude the analysis. We present a simple and efficient PCR strategy to obtain pure bacterial rDNA amplicons from samples predominated by eukaryotic DNA.

Live feed is not a major determinant of the microbiota associated with cod larvae (Gadus morhua)

The gastrointestinal (GI) tract of newly hatched fish is probably colonized by bacteria present in the water, but how environmental and internal factors affect the development of the GI microbiota is poorly understood. In this study, we investigated the effect of diet and of rearing in separate tanks on the cod larval microbiota. Cod larvae were fed three different live feed diets. For each diet, larvae were reared in three replicate tanks. The microbial communities were investigated for water, live feed and individual larvae using a PCR/DGGE (Denaturing Gradient Gel Electrophoresis) strategy. Statistical tests were applied to investigate differences in the larval microbiota between groups of individuals. We found no differences in the larval microbiota due to diet after 8 dph (days post hatching). Moreover, the larval microbiota was similar at 17 and 32 dph, despite a change in live feed at 18 dph. The larval microbiota was generally more similar to the water microbiota than to live feed microbiota. We further found that rearing of larvae in replicate tanks with identical diet could result in significant differences in larval microbiota. These findings indicate that diet does not entail major changes to the composition of cod larval microbiota.

Improved Antifungal Polyene Macrolides via Engineering of the Nystatin Biosynthetic Genes in Streptomyces noursei

Seven polyene macrolides with alterations in the polyol region and exocyclic carboxy group were obtained via genetic engineering of the nystatin biosynthesis genes in Streptomyces noursei. In vitro analyses of the compounds for antifungal and hemolytic activities indicated that combinations of several mutations caused additive improvements in their activity-toxicity properties. The two best analogs selected on the basis of in vitro data were tested for acute toxicity and antifungal activity in a mouse model. Both analogs were shown to be effective against disseminated candidosis, while being considerably less toxic than amphotericin B. To our knowledge, this is the first report on polyene macrolides with improved in vivo pharmacological properties obtained by genetic engineering. These results indicate that the engineered nystatin analogs can be further developed into antifungal drugs for human use.

Different bacterial communities in ectomycorrhizae and surrounding soil

Several eukaryotic symbioses have shown to host a rich diversity of prokaryotes that interact with their hosts. Here, we study bacterial communities associated with ectomycorrhizal root systems of Bistorta vivipara compared to bacterial communities in bulk soil using pyrosequencing of 16S rRNA amplicons. A high richness of Operational Taxonomic Units (OTUs) was found in plant roots (3,571 OTUs) and surrounding soil (3,476 OTUs). The community composition differed markedly between these two environments. Actinobacteria, Armatimonadetes, Chloroflexi and OTUs unclassified at phylum level were significantly more abundant in plant roots than in soil. A large proportion of the OTUs, especially those in plant roots, presented low similarity to Sanger 16S rRNA reference sequences, suggesting novel bacterial diversity in ectomycorrhizae. Furthermore, the bacterial communities of the plant roots were spatially structured up to a distance of 60 cm, which may be explained by bacteria using fungal hyphae as a transport vector. The analyzed ectomycorrhizae presents a distinct microbiome, which likely influence the functioning of the plant-fungus symbiosis.

The expression of recombinant genes in Escherichia coli can be strongly stimulated at the transcript production level by mutating the DNA-region corresponding to the 5-untranslated part of mRNA

Secondary structures and the short Shine–Dalgarno sequence in the 5′‐untranslated region of bacterial mRNAs (UTR) are known to affect gene expression at the level of translation. Here we report the use of random combinatorial DNA sequence libraries to study UTR function, applying the strong, σ32/σ38‐dependent, and positively regulated Pm promoter as a model. All mutations in the libraries are located at least 8 bp downstream of the transcriptional start site. The libraries were screened using the ampicillin‐resistance gene (bla) as reporter, allowing easy identification of UTR mutants that display high levels of expression (up to 20‐fold increase relative to the wild‐type at the protein level). Studies of the two UTR mutants identified by a modified screening procedure showed that their expression is stimulated to a similar extent at both the transcript and protein product levels. For one such mutant a model analysis of the transcription kinetics showed significant evidence of a difference in the transcription rate (about 18‐fold higher than the wild type), while there was no evidence of a difference in transcript stability. The two UTR sequences also stimulated expression from a constitutive σ70‐dependent promoter (P1/Panti‐tet), demonstrating that the UTR at the DNA or RNA level has a hitherto unrecognized role in transcription.

Random Mutagenesis of the Pm Promoter as a Powerful Strategy for Improvement of Recombinant-Gene Expression

ABSTRACT The inducible Pm-xylS promoter system has proven useful for production of recombinant proteins in several gram-negative species and in high-cell-density cultivations of Escherichia coli. In this study we subjected a 24-bp region of Pm (including the −10 element) to random mutagenesis, leading to large mutant libraries in E. coli. Low-frequency-occurring Pm mutants displaying strongly increased promoter activity (up-mutants) could be efficiently identified by using β-lactamase as a reporter. The up-mutants typically carried multiple point mutations positioned throughout the mutagenized region, combined with deletions around the transcription start site. Mutants displaying up to about a 14-fold increase in β-lactamase expression (relative to wild-type Pm) were identified without loss of the inducible phenotype. The mutants also strongly stimulated the expression of two other reporter genes, luc (encoding firefly luciferase) and celB (encoding phosphoglucomutase), and were found to significantly improve (twofold) a previously optimized process for high-level recombinant production of the medically important granulocyte-macrophage colony-stimulating factor in E. coli under high-cell-density conditions. These results demonstrate the potential of using random mutagenesis of promoters to improve protein expression at industrial levels and indicate that targeted modifications of individual functional elements are not sufficient to obtain optimized promoter sequences.

Selection in the host structures the microbiota associated with developing cod larvae (Gadus morhua)

Marine fish larvae are immature upon hatching, and share their environment with high numbers of bacteria. The microbial communities associated with developing fish larvae might be structured by other factors than those important in developing terrestrial animals. Here, we analysed the beta (β)-diversity of the microbiota associated with developing cod larvae and compared it with the bacterial communities in water and live feed by applying pyrosequencing of bar coded v4 16S rDNA amplicons. A total of 15 phyla were observed in the cod larval microbiota. Proteobacteria was the most abundant, followed by Firmicutes, Bacteroidetes and Actinobacteria. The composition and diversity of the cod larval microbiota changed considerably with age. The temporal and spatial patterns of β-diversity could not be explained by stochastic processes, and did not coincide with changes in the rearing conditions. Furthermore, the larval microbiota was highly distinct from the water and the live feed microbiota, particularly at early developmental stages. However, the similarity between larval and water microbiota increased with age. This study suggests that strong selection in the host structures the cod larval microbiota. The changes in community structure observed with increasing age can be explained by altered selection pressure due to development of the intestinal system.

New Nystatin-Related Antifungal Polyene Macrolides with Altered Polyol Region Generated via Biosynthetic Engineering of Streptomyces noursei

ABSTRACT Polyene macrolide antibiotics, including nystatin and amphotericin B, possess fungicidal activity and are being used as antifungal agents to treat both superficial and invasive fungal infections. Due to their toxicity, however, their clinical applications are relatively limited, and new-generation polyene macrolides with an improved therapeutic index are highly desirable. We subjected the polyol region of the heptaene nystatin analogue S44HP to biosynthetic engineering designed to remove and introduce hydroxyl groups in the C-9-C-10 region. This modification strategy involved inactivation of the P450 monooxygenase NysL and the dehydratase domain in module 15 (DH15) of the nystatin polyketide synthase. Subsequently, these modifications were combined with replacement of the exocyclic C-16 carboxyl with the methyl group through inactivation of the P450 monooxygenase NysN. Four new polyene macrolides with up to three chemical modifications were generated, produced at relatively high yields (up to 0.51 g/liter), purified, structurally characterized, and subjected to in vitro assays for antifungal and hemolytic activities. Introduction of a C-9 hydroxyl by DH15 inactivation also blocked NysL-catalyzed C-10 hydroxylation, and these modifications caused a drastic decrease in both antifungal and hemolytic activities of the resulting analogues. In contrast, single removal of the C-10 hydroxyl group by NysL inactivation had only a marginal effect on these activities. Results from the extended antifungal assays strongly suggested that the 9-hydroxy-10-deoxy S44HP analogues became fungistatic rather than fungicidal antibiotics.

Directed evolution of the transcription factor XylS for development of improved expression systems

The inducible Pm promoter together with its cognate positive transcription regulator XylS has been shown to be useful for recombinant protein production under high cell density conditions. Here we report directed evolution of XylS resulting in mutant proteins with increased ability to stimulate transcription in Escherichia coli from Pm. A first round of mutagenesis using error‐prone PCR on xylS was used to construct a library consisting of about 430 000 clones, and this library could be efficiently screened with respect to stimulation of expression from Pm due to a positive correlation between the level of expression of the reporter gene, bla (encoding β‐lactamase), and the ampicillin tolerance of the corresponding host cells. Fourteen different amino acid substitutions in XylS were found to separately lead to up to nearly a threefold stimulation of expression under induced conditions, relative to wild type. These mutations were all located in the part corresponding to the N‐terminal half of the protein. Varying combinations of the mutations resulted in further stimulation, and the best results (about 10‐fold stimulation under induced conditions) were obtained by using a random shuffling procedure followed by a new round of screening. The uninduced levels of expression for the same mutants also increased, but only about four times. Through in silico 3D modelling of the N‐terminal domain of XylS, it was observed that the evolved mutant proteins contained substitutions that were positioned in different parts of the predicted structure, including a β‐barrel putatively responsible for effector binding and a coiled coil probably important for dimerization. The total production of the host‐toxic antibody fragment scFv‐phOx expressed from Pm with the evolved XylS mutant protein StEP‐13 was about ninefold higher than with wild‐type XylS, demonstrating that directed evolution of transcription factors can be an important new tool to achieve high‐level recombinant protein production.

Nitrification at different salinities: Biofilm community composition and physiological plasticity.

This paper describes an experimental study of microbial communities of three moving bed biofilm reactors (MBBR) inoculated with nitrifying cultures originated from environments with different salinity; freshwater, brackish (20‰) and seawater. All reactors were run until they operated at a conversion efficiency of >96%. The microbial communities were profiled using 454-pyrosequencing of 16S rRNA gene amplicons. Statistical analysis was used to investigate the differences in microbial community structure and distribution of the nitrifying populations with different salinity environments. Nonmetric multidimensional scaling analysis (NMDS) and the PERMANOVA test based on Bray-Curtis similarities revealed significantly different community structure in the three reactors. The brackish reactor showed lower diversity index than fresh and seawater reactors. Venn diagram showed that 60 and 78% of the total operational taxonomic units (OTUs) in the ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) guild, respectively, were unique OTUs for a given reactor. Similarity Percentages (SIMPER) analysis showed that two-thirds of the total difference in community structure between the reactors was explained by 10 OTUs, indicating that only a small number of OTUs play a numerically dominant role in the nitrification process. Acute toxicity of salt stress on ammonium and nitrite oxidizing activities showed distinctly different patterns, reaching 97% inhibition of the freshwater reactor for ammonium oxidation rate. In the brackish culture, inhibition was only observed at maximal level of salinity, 32‰. In the fully adapted seawater culture, higher activities were observed at 32‰ than at any of the lower salinities.