Marcin Gołębiewski

Reduced colonic microbial diversity is associated with colitis in NHE3-deficient mice

Chronic inflammation and enteric infections are frequently associated with epithelial Na(+)/H(+) exchange (NHE) inhibition. Alterations in electrolyte transport and in mucosal pH associated with inflammation may represent a key mechanism leading to changes in the intestinal microbial composition. NHE3 expression is essential for the maintenance of the epithelial barrier function. NHE3(-/-) mice develop spontaneous distal chronic colitis and are highly susceptible to dextran sulfate (DSS)-induced mucosal injury. Spontaneous colitis is reduced with broad-spectrum antibiotics treatment, thus highlighting the importance of the microbiota composition in NHE3 deficiency-mediated colitis. We herein characterized the colonic microbiome of wild-type (WT) and NHE3(-/-) mice housed in a conventional environment using 454 pyrosequencing. We demonstrated a significant decrease in the phylogenetic diversity of the luminal and mucosal microbiota of conventional NHE3(-/-) mice compared with WT. Rederivation of NHE3(-/-) mice from conventional to a barrier facility eliminated the signs of colitis and decreased DSS susceptibility. Reintroduction of the conventional microflora into WT and NHE3(-/-) mice from the barrier facility resulted in the restoration of the symptoms initially described in the conventional environment. Interestingly, qPCR analysis of the microbiota composition in mice kept in the barrier facility compared with reconventionalized mice showed a significant reduction of Clostridia classes IV and XIVa. Therefore, the gut microbiome plays a prominent role in the pathogenesis of colitis in NHE3(-/-) mice, and, reciprocally, NHE3 also plays a critical role in shaping the gut microbiota. NHE3 deficiency may be a critical contributor to dysbiosis observed in patients with inflammatory bowel disease.

Adaptative potential of the Lactococcus lactis IL594 strain encoded in its 7 plasmids.

The extrachromosomal gene pool plays a significant role both in evolution and in the environmental adaptation of bacteria. The L. lactis subsp. lactis IL594 strain contains seven plasmids, named pIL1 to pIL7, and is the parental strain of the plasmid-free L. lactis IL1403, which is one of the best characterized lactococcal strains of LAB. Complete nucleotide sequences of pIL1 (6,382 bp), pIL2 (8,277 bp), pIL3 (19,244 bp), pIL4 (48,979), pIL5 (23,395), pIL6 (28,435 bp) and pIL7 (28,546) were established and deposited in the generally accessible database (GeneBank). Nine highly homologous repB-containing replicons, belonging to the lactococcal theta-type replicons, have been identified on the seven plasmids. Moreover, a putative region involved in conjugative plasmid mobilization was found on four plasmids, through identification of the presence of mob genes and/or oriT sequences. Detailed bioinformatic analysis of the plasmid nucleotide sequences provided new insight into the repertoire of plasmid-encoded functions in L. lactis, and indicated that plasmid genes from IL594 strain can be important for L. lactis adaptation to specific environmental conditions (e.g. genes coding for proteins involved in DNA repair or cold shock response) as well as for technological processes (e.g. genes encoding citrate and lactose utilization, oligopeptide transport, restriction-modification system). Moreover, global gene analysis indicated cooperation between plasmid- and chromosome-encoded metabolic pathways.

Analysis of the complete genome sequence of the lactococcal bacteriophage bIBB29

Bacteriophage bIBB29 was isolated from a whey sample originating from an industrial biotechnological process, disturbed by a bacteriophage attack. Phage bIBB29 was determined to be active against three phage-resistant strains of Lactococcus lactis. It belongs to the 936 species containing virulent phages with isometric head and short non-contractile tail. One-step growth kinetics of bIBB29 phage showed that its latent time was 23 min, and the burst size was about 130 bacteriophages. The complete nucleotide sequence of the virulent L. lactis bacteriophage bIBB29 comprises 29305 nucleotides and is the sixth phage genome of the 936 species published until now. The G+C content of the bIBB29 genome (34.7%) is similar to that of its host and also to that of other phages from the 936 species. The bIBB29 genome counts 54 open reading frames organized in three typical clusters, corresponding to the early, middle and late expressed genes. Only 20 protein products of the predicted genes were found to have their homologs among proteins with known function. The early expressed region in the genomes of 936 group members displays the highest divergence, whereas the late and middle regions share high similarities, with the exception of five genes. The genome of bIBB29 shares the highest overall nucleotide similarity with bIL170 (87%), and the lowest with phage 712 (77%). The host range analysis showed that despite the high level of similarity between the receptor binding protein (RBP) of phage bIBB29 and P475, they have a different host range. This implies that RBP is not a sufficient factor for host range.

Transcriptomic profiling of the salt stress response in excised leaves of the halophyte Beta vulgaris ssp. maritima.

Beta vulgaris ssp. maritima is a halophytic relative of cultivated beets. In the present work a transcriptome response to acute salt stress imposed to excised leaves of sea beet was investigated. Salt treatments consisted of adding NaCl directly to the transpiration stream by immersing the petioles of excised leaves into the salt solutions. Sequencing libraries were generated from leaves subjected to either moderate or strong salt stress. Control libraries were constructed from untreated leaves. Sequencing was performed using the Illumina MiSeq platform. We obtained 32970 unigenes by assembling the pooled reads from all the libraries with Trinity software. Screening the nr database returned 18,362 sequences with functional annotation. Using the reference transcriptome we identified 1,246 genes that were differentially expressed after 48 h of NaCl stress. Genes related to several cellular functions such as membrane transport, osmoprotection, molecular chaperoning, redox metabolism or protein synthesis were differentially expressed in response to salt stress. The response of sea beet leaves to salt treatments was marked out by transcriptomic up-regulation of genes related to photosynthetic carbon fixation, ribosome biogenesis, cell wall-building and cell wall expansion. Furthermore, several novel and undescribed transcripts were responsive to salinity in leaves of sea beet.

Tideless estuaries in brackish seas as a possible freshwater-marine transition zones for bacteria – the case study of the Vistula river estuary

Most bacteria are found either in marine or fresh waters and transitions between the two habitats are rare, even though freshwater and marine bacteria co-occur in brackish habitats. Estuaries in brackish, tideless seas could be habitats where the transition of freshwater phylotypes to marine conditions occurs. We tested this hypothesis in the Gulf of Gdańsk (Baltic Sea) by comparing bacterial communities from different zones of the estuary, via pyrosequencing of 16S rRNA amplicons. We predicted the existence of a core microbiome (CM, a set of abundant OTUs present in all samples) comprising OTUs consisting of populations specific for particular zones of the estuary. The CMs for the entire studied period consisted of only eight OTUs, and this number was even lower for specific seasons: five in spring, two in summer, and one in autumn and winter. Six of the CM OTUs, and another 21 of the 50 most abundant OTUs consisted of zone-specific populations, plausibly representing micro-evolutionary forces. The presence of up to 15% of freshwater phylotypes from the Vistula River in the brackish Gulf of Gdańsk supported our hypothesis, but high dissimilarity between the bacterial communities suggested that freshwater-marine transitions are rare even in tideless estuaries in brackish seas.

Rapid Microbial Community Changes During Initial Stages of Pine Litter Decomposition

Plant litter decomposition is a process enabling biogeochemical cycles closing in ecosystems, and decomposition in forests constitutes the largest part of this process taking place in terrestrial biomes. Microbial communities during litter decomposition were studied mainly with low-throughput techniques not allowing detailed insight, particularly into coniferous litter, as it is more difficult to obtain high quality DNA required for analyses. Motivated by these problems, we analyzed archaeal, bacterial, and eukaryotic communities at three decomposition stages: fresh, 3- and 8-month-old litter by 16/18S rDNA pyrosequencing, aiming at detailed insight into early stages of pine litter decomposition. Archaea were absent from our libraries. Bacterial and eukaryotic diversity was greatest in 8-month-old litter and the same applied to bacterial and fungal rDNA content. Community structure was different at various stages of decomposition, and phyllospheric organisms (bacteria: Acetobacteraceae and Pseudomonadaceae members, fungi: Lophodermium, Phoma) were replaced by communities with metabolic capabilities adapted to the particular stage of decomposition. Sphingomonadaceae and Xanthomonadaceae and fungal genera Sistotrema, Ceuthospora, and Athelia were characteristic for 3-month-old samples, while 8-month-old ones were characterized by Bradyrhizobiaceae and nematodes (Plectus). We suggest that bacterial and eukaryotic decomposer communities change at different stages of pine litter decomposition in a way similar to that in broadleaf litter. Interactions between bacteria and eukaryotes appear to be one of the key drivers of microbial community structure.

How Does Salinity Shape Bacterial and Fungal Microbiomes of Alnus glutinosa Roots

Black alder (Alnus glutinosa Gaertn.) belongs to dual mycorrhizal trees, forming ectomycorrhizal (EM) and arbuscular (AM) root structures, as well as represents actinorrhizal plants that associate with nitrogen-fixing actinomycete Frankia sp. We hypothesized that the unique ternary structure of symbionts can influence community structure of other plant-associated microorganisms (bacterial and fungal endophytes), particularly under seasonally changing salinity in A. glutinosa roots. In our study we analyzed black alder root bacterial and fungal microbiome present at two forest test sites (saline and non-saline) in two different seasons (spring and fall). The dominant type of root microsymbionts of alder were ectomycorrhizal fungi, whose distribution depended on site (salinity): Tomentella, Lactarius, and Phialocephala were more abundant at the saline site. Mortierella and Naucoria (representatives of saprotrophs or endophytes) displayed the opposite tendency. Arbuscular mycorrhizal fungi belonged to Glomeromycota (orders Paraglomales and Glomales), however, they represented less than 1% of all identified fungi. Bacterial community structure depended on test site but not on season. Sequences affiliated with Rhodanobacter, Granulicella, and Sphingomonas dominated at the saline site, while Bradyrhizobium and Rhizobium were more abundant at the non-saline site. Moreover, genus Frankia was observed only at the saline site. In conclusion, bacterial and fungal community structure of alder root microsymbionts and endophytes depends on five soil chemical parameters: salinity, phosphorus, pH, saturation percentage (SP) as well as total organic carbon (TOC), and seasonality does not appear to be an important factor shaping microbial communities. Ectomycorrhizal fungi are the most abundant symbionts of mature alders growing in saline soils. However, specific distribution of nitrogen-fixing Frankia (forming root nodules) and association of arbuscular fungi at early stages of plant development should be taken into account in further studies.