Polyphasic taxonomic studies of lactic acid bacteria associated with Tunisian fermented meat based on the heterogeneity of the 16S–23S rRNA gene intergenic spacer region

The objective of this work was to investigate the structure and diversity of lactic acid bacteria (LAB) communities in traditionally fermented meat collected from different areas of Tunisia. A polyphasic study, which involves phenotypic tests and ribosomal DNA-based techniques, was used to identify Gram-positive and catalase-negative isolates. PCR amplification of the 16S–23S rDNA ISR of 102 isolates and other reference LAB strains gave (1) one type of rrn operon (M-ISR) for lactococci, (2) two types of rrn operon (S-ISR and M-ISR) for enterococci, (3) two types of rrn operon (S-ISR and L-ISR) for Lactobacilli, and (4) three PCR amplicons (S-ISR, M-ISR, and L-ISR) obtained for Pediococcus spp. and Weissella genus. The clustering and comparison of ISR–RFLP profiles given by the isolates with those given by reference LAB strains, allowed their identification as Lactococcus lactis, Enterococcus faecium, Enterococcus faecalis, Enterococcus sanguinicola, Enterococcus hawaiiensis, Lactobacillus sakei, Lactobacillus curvatus, Lactobacillus plantarum, Lactobacillus alimentarius, Pediococcus pentosaceus, and Weissella confusa. Combined 16S–23S rDNA ISR and RFLP patterns can be considered as a good potential target for a rapid and reliable differentiation between isolates of LAB and provided further information on the organization of their rrn operons.     

Analysis of the Gut Microbiota by High-Throughput Sequencing of the V5–V6 Regions of the 16S rRNA Gene in Donkey

Considerable evidence suggests that the gut microbiota is complex in many mammals and gut bacteria communities are essential for maintaining gut homeostasis. To date the research on the gut microbiota of donkey is surprisingly scarce. Therefore, we performed high-throughput sequencing of the 16S rRNA genes V5–V6 hypervariable regions from gut fecal material to characterize the gut microbiota of healthy donkeys and compare the difference of gut microbiota between male and female donkeys. Sixty healthy donkeys (30 males and 30 females) were enrolled in the study, a total of 915,691 validated reads were obtained, and the bacteria found belonged to 21 phyla and 183 genera. At the phylum level, the bacterial community composition was similar for the male and female donkeys and predominated by Firmicutes (64 % males and 64 % females) and Bacteroidetes (23 % males and 21 % females), followed by Verrucomicrobia, Euryarchaeota, Spirochaetes, and Proteobacteria. At the genus level, Akkermansia was the most abundant genus (23 % males and 17 % females), followed by Sporobacter, Methanobrevibacter, and Treponema, detected in higher distribution proportion in males than in females. On the contrary, Acinetobacter and Lysinibacillus were lower in males than in females. In addition, six phyla and 15 genera were significantly different between the male and female donkeys for species abundance. These findings provide previously unknown information about the gut microbiota of donkeys and also provide a foundation for future investigations of gut bacterial factors that may influence the development and progression of gastrointestinal disease in donkey and other animals.     

Effects of neutrophils peptide-1 transgenic <Emphasis Type="Italic">Chlorella ellipsoidea</Emphasis> on the gut microbiota of male Sprague–Dawley rats,as revealed by high-throughput 16S rRNA sequencing

Rabbit neutrophils peptide-1 (NP-1) is a type of defensin that possesses a broad spectrum of antimicrobial activity. Chlorella ellipsoidea is a new eukaryotic expression system for exogenously producing NP-1. The NP-1 transgenic C. ellipsoidea can be directly added into feed as antimicrobial agent without any purification procedure for the NP-1 peptide. However, the effects of C. ellipsoidea and NP-1 on the host gut microbiota should be explored before application. In this study, diets containing different concentrations (1.25, 2.5, and 5 %) of C. ellipsoidea and NP-1 transgenic C. ellipsoidea were administered to male Sprague–Dawley rats. Compared with the chow diet control group, none of the experimental groups showed any significant differences in their growth indices, and no histopathological damage was observed. The phylotypes of gut microbiota in the control group, the 5 % C. ellipsoidea diet group and the 5 % NP-1 transgenic C. ellipsoidea diet group were determined by 16S rRNA sequencing. The results showed that both 5 % experimental groups had shifted community memberships of gut microbiota. In particular, the 5 % NP-1 transgenic C. ellipsoidea diet exhibited an increased abundance of most Gram-positive bacterial taxa and a reduced abundance of most Gram-negative bacterial taxa, and it promoted the growth of some lactic acid bacterial genera. Lactic acid bacteria, especially the Bifidobacterium and Lactobacillus, have been widely reported to be benefic effects on the host. Thus NP-1 transgenic C. ellipsoidea is promising feed additive and gut regulator, as it have the potential to increase the abundance of Bifidobacterium and Lactobacillus in gut microbiota of animal.     

Sequence organization of the chloroplast ribosomal spacer ofSpinacia oleracea including the 3′ end of the 16S rRNA and the 5′ end of the 23S rRNA

The 2201-bp spacer between the chloroplast ribosomal 16S and 23S genes ofSpinacia oleracea was sequenced. It contains the genes of the tRNA^Ile (GAU) and tRNA^Ala (UGC) which are both interrupted by introns of respectively 728 and 816 bp. These introns belong to the class II according to the classfication of Michel and Dujon [17]. Comparison of the rDNA spacer sequence of maize, tobacco and spinach indicates that no conserved polypeptide is encoded within the introns of the two tRNA genes and that the two main insertions/deletions between the three plants are located within two loops of the class II introns secondary structure, which is therefore conserved. Based on the sequence complementarity observed between the upstream and downstream parts, of the 16S and 23S rRNA genes, RNase III-like secondary structures involved in the processing of the rRNA precursor are proposed.     

Assessment of the modulating effects of fructo-oligosaccharides on fecal microbiota using human flora–associated piglets

We first used human flora–associated (HFA) piglets, a significantly improved model for research on human gut microbiota, to study the effects of short-chain fructo-oligosaccharides (scFOS) on the gut bacterial populations. Ten neonatal HFA piglets were assigned to receive basal diets alone or supplemented with scFOS (0.5 g/kg body weight/day) from 1 to 37 days after birth (DAB). The impact of scFOS on the fecal bacterial populations of the piglets before (12 DAB), during (17 DAB), and after (25 and 37 DAB) weaning were monitored by PCR-denaturing gradient gel electrophoresis and real-time quantitative PCR. The Bifidobacterium genus was stimulated consistently, except during weaning, confirming the bifidogenic property of scFOS. At 12 DAB, the Clostridium leptum subgroup was decreased and two unknown Bacteroides-related species were increased; at 25 DAB, the C. leptum subgroup and Subdoligranulum variabile-like species were elevated, whereas one unknown Faecalibacterium-related species was suppressed; and at 37 DAB, the Bacteroides genus was decreased. The results showed that effects of scFOS on non-bifidobacteria varied at different developmental stages of the animals, warranting further investigation into the host-development-related effects of prebiotics on the gut microbiota and the host physiology using the HFA piglets as a model for humans.     

Phylogenetic relationship of 16 Oedipodidae species （Insecta： Orthoptera） based on the 16S rRNA gene sequences

The sequences of the mitochondrial 16S rRNA gene of 16 Oedipodidae species were amplified and sequenced. All sequences were aligned and analyzed and the phylogenetic relationships were inferred. The properties of 16S gene in Oedipodidae showed typical patterns of many insects such as a high A＋T content and variable distance-dependent transition/transversion ratios. The 0.2 weight for sites of loops may be advisable for phylogeny reconstruction using the maximum parsimony method. The phylogenetic analysis results do not support the current subfamily classification systems of Oedipodidae. Bryodemellinae and Bryodeminae are closely related and should be merged as one subfamily. The status of Oedipodinae and Locustinae is also problematic.     

16S–23S rRNA Intergenic Spacer Region Variability in the Genus Frankia

16S–23S rRNA internally transcribed spacer (ITS) sequences from 53 Frankia strains were sequenced and sized from polymerase chain reaction amplification products and compiled with 14 selected 16S–23S ITS sequences from public database. Frankia genomes included two to three ITS copies lacking length polymorphism except for nine strains. No tRNA gene was encountered in this region. Frankia strains exhibited various lengths (369 to 452 nt) and a wide range of sequence similarity (35–100%) in the ITS region. The average pairwise distance varied from 0.368 (clusters 1 and 2) to 0.964 (clusters 3 and 4) and was 0.397, 0.138, 0.129, and 0.016, respectively, for cluster 4 (saprophytic non-infective/non-effective), clusters 1 and 3 (facultative symbiotic), and cluster 2 (obligate symbiotic). This suggests a gradual erosion of Frankia diversity concomitantly with a shift from saprophytic non-infective/non-effective to facultative and symbiotic lifestyle. Comparative sequence analyses of the 16S–23S rRNA intergenic spacer region of Frankia strains are not useful to assign them to their respective cluster or host infection group. Accurate assignment required the inclusion of the adjacent 16S and 23S rRNA gene fragments.     

Sequence studies on the soybean chloroplast 16S–23S rDNA spacer region

The sequence of the ribosomal spacer region of soybean chloroplast DNA including the 3 end of the 16S rRNA gene, the tRNA^Ala and tRNA^Ile genes (but not their introns), the three intergenic regions and the 5 end of the 23S rRNA gene, has been determined. This sequence has been compared to corresponding regions of other angiosperm chloroplast DNAs. Secondary structure models are proposed for the entirety of the intergenic regions a, b and c and for the flanking rRNA regions. A model for a common secondary structure of the ribosomal spacer intergenic regions from chloroplasts of higher plants is proposed, which is supported by comparative evidence.     

Differentiation of non-pylori Helicobacter species based on PCR–restriction fragment length polymorphism of the 23S rRNA gene

Phenotypic identification of non-pylori Helicobacter species has always been problematic and time-consuming in comparison with many other bacteria. We developed a rapid two-step identification assay based on PCR–restriction fragment length polymorphism (PCR–RFLP) analysis of the 23S rRNA gene for differentiating between non-pylori Helicobacter species. A new genus-specific primer pair based on all available complete and partial 23S rRNA sequences of Helicobacter species was designed. In silico restriction analysis of variable regions of the 23S rRNA gene suggested SmaI and HindIII endonucleases would provide a good level of differentiation. Analysis of the obtained 23S rRNA RFLP patterns divided all Helicobacter study strains into three species groups (groups A–C) and 12 unique restriction patterns. Wolinella succinogenes also gave a unique pattern. Our proposed PCR–RFLP method was found to be as a valuable tool for routine identification of non-pylori Helicobacter species from human or animal samples.     

16S rRNA基因高变区V4和V3−V4及测序深度对油藏细菌菌群分析的影响

【背景】16S rRNA基因测序是当前研究微生物群落组成及其分布的重要手段。【目的】揭示16S rRNA基因高变区V4 (515-806)和V3-V4 (338-806)及测序深度(1-2万条和10万条)对油藏微生物细菌群落组成和多样性分析的影响。【方法】所用油水样细菌16SrRNA基因拷贝数为(6.51±0.56)×108/L,16SrRNA基因V4区测序使用IlluminaMiSeqPE250测序平台,V3-V4区测序使用MiSeqPE300测序平台。【结果】测序深度达到1-2万条时,V4和V3-V4区测序文库覆盖率均达到99.6%以上,且具有较好的可重复性;V4区测序深度为1-2万和10万时,菌群α多样性指数受测序深度影响不显著;与V4区测序相比,同样测序深度(1-2万)下,V3-V4区测序获得的菌群α多样性指数有所降低。V4测序1-2万与10万获得的菌群中几乎未出现显著性差异微生物类群;同样测序深度(1-2万)下,V4与V3-V4测序相比,优势微生物类群Epsilonproteobacteria(51.37%:64.23%)和Deltaproteobacteria (17.96%:11.40%)相对丰度表现出显著差异。【结论】测序深度达到一定水平,增加测序深度会一定程度上影响菌群α多样性指数,对菌群β多样性分析的影响十分有限;同一测序深度下,V4区与V3-V4区测序获得的细菌菌群α多样性指数明显不同,部分优势微生物类群的相对丰度值之间具有显著性差异。鉴于测序读长的提升和测序成本降低,与V4区测序相比,V3-V4区测序在更低的测序深度下文库覆盖率更高,可提供更多用于反映物种亲缘关系的16S rRNA碱基信息,本文认为V3-V4区测序可作为当下菌群分析的首选区域。     

Ribosomal RNA genes ofEndomyces fibuliger: isolation, sequencing and the use of the 26S rRNA gene in integrative transformation ofSaccharomyces cerevisiae for efficient expression of the α-amylase gene ofEndomyces fibuliger

Endomyces fibuliger is a dimorphic yeast which is homothallic and exists predominantly in the diploid phase with a brief haploid phase. A repeat unit of the ribosomal RNA genes, or rDNA, from E. fibuliger 8014 met has been isolated, cloned and sequenced. In this report, the sequences of the 17S, 5.8S and 26S rRNA genes are presented. Homology between the sequenced rRNA genes and those of closely-related yeast strains, particularly Saccharomyes cerevisiae and Candida albicans, was observed. As a step towards the eventual development of a transformation system for the yeast E. fibuliger, an integrative plasmid containing the 5.8S and a part of the 26S rRNA gene, a selectable marker conferring resistance to the G418 antibiotic and a reporter gene, the α-amylase (ALP1) gene of E. fibuliger, was constructed. This plasmid was linearized at a unique restriction site within the 26S rRNA gene, and transformed into S. cerevisiae INVSC2 MATa his3 ura3 using the lithium acetate method to test the functionality of the vector system. Transformation into S. cerevisiae INVSC2 MATa his3 ura3 was by virtue of the extensive homology between the sequenced 26S rRNA gene of E. fibuliger 8014 met and that of S. cerevisiae, so that homologous pairing and integration into the recipient chromosome was possible. The G418-resistant S. cerevisiae transformants produced halos on starch medium due to hydrolysis by α-amylase, and they were further analysed by Southern hybridization with the ALP1 gene and the gene encoding the aminoglycoside 3′- phosphotransferase I enzyme which confers resistance to the G418 antibiotic. A band of 13.7 kb which corresponded to the linearized size of the transforming plasmid DNA was obtained on the autoradiogram, suggesting that tandem copies of the plasmid DNA are present in the chromosome. Finally, an assay of the α-amylase enzyme secreted extracellularly was performed on the transformants.     

Identification of hypervariable regions within the 16S–23S rRNA intergenic spacer region of Flavobacterium columnare and its application in assigning genomovar group to an individual strain

Flavobacterium columnare is an important bacterial pathogen of fish with a wide genetic variability within the species. This intra-species diversity has been termed as genomovars and genomovar groups on the basis of Restriction Fragment Length Polymorphisms of 16S rDNA and 16S–23S rDNA intergenic spacer region (ISR), respectively. In this study, we demonstrate the source of genetic heterogeneity in the F. columnare by sequence analysis of ISR. The length of ISR sequences of different genomovars varied from 553 to 592 nucleotides, while the similarity among sequences ranged from 76.1 to 92.6%. A common ISR structure with tRNA^Ala and tRNA^Ile embedded within the sequence was identified in all the genomovars of F. columnare. The results show that strains of F. columnare can be categorized into five genomovar groups based on the heterogeneity of the ISR sequences. Of these, strains belonging to Genomovar I and II can be sub-divided into two groups each; while strains of Genomovar III belong to one group. Sequence similarity between genomovar groups was lower for ISR (76.1–92.6%) as compared to 16S rDNA (96.1–99.4%) indicating its ability to resolve closely related groups within the genomovars of F. columnare. The main source of variation between the genomovar groups is the presence of three hyper variable regions (V1, V2, and V3) in the ISR. Of the three, V3 was found to be the most heterogeneous region and was found to be useful in assigning a genomovar group to an individual strain of F. columnare.     

Expression of the glutathione peroxidase gene lacking its 3′ untranslated region

In order to investigate the expression of human cellular glutathione peroxidase (GPx), we mutated the gene encoding GPx by deleting either the 5 or 3 untranslated region (utr), subcloned the deleted fragments into plasmid pSVL followed by transfection into COS-7 cells and measured the amount of GPx expressed. When the 5 utr of the gene was deleted, GPx was not expressed. However, the deletion of the 3 utr resulted in some expression of GPx. Deletion of the poly A region of the GPx gene resulted in the expression of GPx but the level was lower than that of the full-length cGPx. The complete deletion of the 3 utr resulted in a half of the expression of the poly A deletion mutant. Thus, the expression of GPx increased according to the length of the 3 utr. These results suggest that the GPx gene carrying one SECIS on 5 utr (FEBS Lett. 312(1992)10-14) is essential for GPx expression. SECIS on 3 utr might not play a key role of GPx expression. Expression of GPx by COS-7 cells was not observed when a plasmid harboring an antisense gene was transfected.     

Localization of the human insulin-like growth-factor-binding protein 4 gene to chromosomal region 17q12–21.1

Summary Insulin-like growth-factor-binding proteins (IGFBPs) constitute a family of structurally related proteins that specifically bind insulin-like growth factors and modulate their functions. In this study, the chromosomal localization was determined for the gene encoding IGFBP4, i.e. inhibitory-IGFBP. A polymerase chain reaction (PCR) fragment corresponding to the previously published cDNA sequence was used to isolate overlapping cosmid clones. By fluorescent in situ hybridization to metaphase chromosomes, the IGFBP4 gene was then localized to chromosomal region 17q21–21.1. This result was in agreement with PCR analysis of a panel of somatic cell hybrids.     

Complete cloning of the chloroplast genome of safflower in λ EMBL3 and mapping of 23S and 16S rRNA genes

Summary A recombinant DNA library was constructed from partial BamHI or MboI digests of safflower (Carthamus tinctorius L.) chloroplast DNA, in the BamHI site of EMBL3. Seventeen recombinants, selected by chromosome walking, were found to contain overlapping fragments of the entire chloroplast genome. These clones were mapped using single and double digests of BamHI, EcoRI and HindIII. cDNAs synthesized from isolated 16S and 23S chloroplast rRNAs were used to map the ribosomal RNA genes relative to physical maps of the above restriction enzymes. The mapped positions of the rRNA genes for the safflower chloroplast DNA are in good agreement with previously published data for tobacco, spinach and several other higher plants.