CRISPRs of Enterococcus faecalis and E. hirae Isolates from Pig Feces Have Species-Specific Repeats But Share Some Common Spacer Sequences

Clustered regularly interspaced short palindromic repeats (CRISPR) are currently a topic of interest in microbiology due to their role as a prokaryotic immune system. Investigations of CRISPR distribution and characterization to date have focused on pathogenic bacteria, while less is known about CRISPR in commensal bacteria, where they may have a significant role in the ecology of the microbiota of humans and other animals, and act as a recorder of interactions between bacteria and viruses. A combination of PCR and sequencing was used to determine prevalence and distribution of CRISPR arrays in Enterococcus faecalis and Enterococcus hirae isolates from the feces of healthy pigs. Both type II CRISPR–Cas and Orphan CRISPR (without Cas genes) were detected in the 195 isolates examined. CRISPR–Cas was detected in 52 (46/88) and 42 % (45/107) E. faecalis and E. hirae isolates, respectively. The prevalence of Orphan CRISPR arrays was higher in E. faecalis isolates (95 %, 84/88) compared with E. hirae isolates (49 %, 53/107). Species-specific repeat sequences were identified in Orphan CRISPR arrays, and 42 unique spacer sequences were identified. Only two spacers matched previously characterized pig virome sequences, and many were apparently derived from chromosomal sequences of enterococci. Surprisingly, 17 (40 %) of the spacers were detected in both species. Shared spacer sequences are evidence of a lack of species specificity in the agents and mechanisms responsible for integration of spacers, and the abundance of spacer sequences corresponding to bacterial chromosomal sequences reflects interspecific interactions within the intestinal microbiota.     

The postradiation use of vitamin-containing complexes and a phycocyanin extract in a radiation lesion in rats

Wistar rats have been exposed to X-rays with a dose of 5 Gy. Significant decrease in dehydrogenase activity, energy-rich phosphate level and efficiency of antioxidant defence and significant increase in pyruvate amount were observed within 4 weeks. It was also found that the feeding of exposed rats with phycocyanin extract from blue-green algae Spirulina platensis lead to correcting effect. The same result was observed after injections of tocopherol or complex of six water-soluble vitamins. The combination of above mentioned compounds had more marked effect, especially at the presence unitiole and Na2Se.     

Structures of mitochondrial oxidative phosphorylation supercomplexes and mechanisms for their stabilisation

Oxidative phosphorylation (OXPHOS) is the main source of energy in eukaryotic cells. This process is performed by means of electron flow between four enzymes, of which three are proton pumps, in the inner mitochondrial membrane. The energy accumulated in the proton gradient over the inner membrane is utilized for ATP synthesis by a fifth OXPHOS complex, ATP synthase. Four of the OXPHOS protein complexes associate into stable entities called respiratory supercomplexes. This review summarises the current view on the arrangement of the electron transport chain in mitochondrial cristae. The functional role of the supramolecular organisation of the OXPHOS system and the factors that stabilise such organisation are highlighted. This article is part of a Special Issue entitled: Dynamic and ultrastructure of bioenergetic membranes and their components.     

Detection and quantification of 14 <Emphasis Type="Italic">Campylobacter</Emphasis> species in pet dogs reveals an increase in species richness in feces of diarrheic animals

Background  

The genus Campylobacter includes many species, some of which are known human and animal pathogens. Even though studies have repeatedly identified domestic dogs as a risk factor for human campylobacteriosis, our understanding of Campylobacter ecology in this reservoir is limited. Work to date has focused primarily on a limited number of species using culture-based methods. To expand our understanding of Campylobacter ecology in dogs, a collection of fecal samples from 70 healthy and 65 diarrheic pet dogs were examined for the presence and levels of 14 Campylobacter species using quantitative PCR.     

Identification of genes involved in the biosynthesis and attachment of Methanococcus voltae N-linked glycans: insight into N-linked glycosylation pathways in Archaea

N-linked glycosylation is recognized as an important post-translational modification across all three domains of life. However, the understanding of the genetic pathways for the assembly and attachment of N-linked glycans in eukaryotic and bacterial systems far outweighs the knowledge of comparable processes in Archaea. The recent characterization of a novel trisaccharide [beta-ManpNAcA6Thr-(1-4)-beta-GlcpNAc3NAcA-(1-3)-beta-GlcpNAc]N-linked to asparagine residues in Methanococcus voltae flagellin and S-layer proteins affords new opportunities to investigate N-linked glycosylation pathways in Archaea. In this contribution, the insertional inactivation of several candidate genes within the M. voltae genome and their resulting effects on flagellin and S-layer glycosylation are reported. Two of the candidate genes were shown to have effects on flagellin and S-layer protein molecular mass and N-linked glycan structure. Further examination revealed inactivation of either of these two genes also had effects on flagella assembly. These genes, designated agl (archaeal glycosylation) genes, include a glycosyl transferase (aglA) involved in the attachment of the terminal sugar to the glycan and an STT3 oligosaccharyl transferase homologue (aglB) involved in the transfer of the complete glycan to the flagellin and S-layer proteins. These findings document the first experimental evidence for genes involved in any glycosylation process within the domain Archaea.     

A Molecular Enrichment Strategy Based on <Emphasis Type="Italic">cpn</Emphasis>60 for Detection of Epsilon-Proteobacteria in the Dog Fecal Microbiome

Members of the rare microbiome can be important components of complex microbial communities. For example, pet dog ownership is a known risk factor for human campylobacteriosis, and Campylobacter is commonly detected in dog feces by targeted assays. However, these organisms have not been detected by metagenomic methods. The goal of this study was to characterize fecal microbiota from healthy and diarrheic pet dogs using two different levels of molecular detection. PCR amplification and pyrosequencing of the universal cpn60 gene target was used to obtain microbial profiles from each dog. To investigate the relatively rare epsilon-proteobacteria component of the microbiome, a molecular enrichment was carried out using a PCR that first amplified the cpn10–cpn60 region from epsilon-proteobacteria, followed by universal cpn60 target amplification and pyrosequencing. From the non-enriched survey, the major finding was a significantly higher proportion of Bacteroidetes, notably Bacteroides vulgatus, in healthy dogs compared to diarrheic dogs. Epsilon-proteobacteria from the genera Helicobacter and Campylobacter were also detected at a low level in the non-enriched profiles of some dogs. Molecular enrichment increased the proportion of epsilon-proteobacteria sequences detected from each dog, as well as identified novel, presumably rare sequences not seen in the non-enriched profiles. Enriched profiles contained known species of Arcobacter, Campylobacter, Flexispira, and Helicobacter and identified two possibly novel species. These findings add to our understanding of the canine fecal microbiome in general, the epsilon-proteobacteria component specifically, and present a novel modification to traditional metagenomic approaches for study of the rare microbiome.     

Avian predation on a parasitic fly of cervids during winter: can host‐related cues increase the predation risk?

The deer ked (Lipoptena cervi) is an ectoparasitic fly on cervids that has expanded its distribution rapidly in Northern Europe. However, the regulating biotic factors such as predation remain unknown. The host‐independent pupal stage of the fly lasts for several months. Blackish pupae are visible against snow, especially on the bedding sites of hosts, and are thus exposed to predators. To evaluate the role of predation on the invasion dynamics and evolution of L. cervi, we monitored pupal predation on artificial bedding sites in three geographical areas in Finland during winter. We explored: (1) possible predators; (2) magnitude of predation; and (3) whether predation risk is affected by host‐derived cues. We demonstrate that pupae are predated by a number of tit species. Any reddish brown snow discoloration on bedding sites, indicating heavy infestation of the host, serves as an exploitable cue for avian predators, thereby increasing the risk of pupal predation. The ability of tits to use this host‐derived cue seems to be dependent on the prevalence of L. cervi and the period of invasion history, which suggests that it may be a learned behavioural response. Predation by tits may potentially affect the L. cervi population dynamics locally. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 106 , 275–286.     

Identification of a putative acetyltransferase gene, MMP0350, which affects proper assembly of both flagella and pili in the archaeon Methanococcus maripaludis

Glycosylation is a posttranslational modification utilized in all three domains of life. Compared to eukaryotic and bacterial systems, knowledge of the archaeal processes involved in glycosylation is limited. Recently, Methanococcus voltae flagellin proteins were found to have an N-linked trisaccharide necessary for proper flagellum assembly. Current analysis by mass spectrometry of Methanococcus maripaludis flagellin proteins also indicated the attachment of an N-glycan containing acetylated sugars. To identify genes involved in sugar biosynthesis in M. maripaludis, a putative acetyltransferase was targeted for in-frame deletion. Deletion of this gene (MMP0350) resulted in a flagellin molecular mass shift to a size comparable to that expected for underglycosylated or completely nonglycoslyated flagellins, as determined by immunoblotting. Assembled flagellar filaments were not observed by electron microscopy. Interestingly, the deletion also resulted in defective pilus anchoring. Mutant cells with a deletion of MMP0350 had very few, if any, pili attached to the cell surface compared to a nonflagellated but piliated strain. However, pili were obtained from culture supernatants of this strain, indicating that the defect was not in pilus assembly but in stable attachment to the cell surface. Complementation of MMP0350 on a plasmid restored pilus attachment, but it was unable to restore flagellation, likely because the mutant ceased to make detectable flagellin. These findings represent the first report of a biosynthetic gene involved in flagellin glycosylation in archaea. Also, it is the first gene to be associated with pili, linking flagellum and pilus structure and assembly through posttranslational modifications.