Routine phenotypic identification of bacterial species of the family Pasteurellaceae isolated from animals

Pasteurellaceae are bacteria with an important role as primary or opportunistic, mainly respiratory, pathogens in domestic and wild animals. Some species of Pasteurellaceae cause severe diseases with high economic losses in commercial animal husbandry and are of great diagnostic concern. Because of new data on the phylogeny of Pasteurellaceae, their taxonomy has recently been revised profoundly, thus requiring an improved phenotypic differentiation procedure to identify the individual species of this family. A new and simplified procedure to identify species of Actinobacillus, Avibacterium, Gallibacterium, Haemophilus, Mannheimia, Nicoletella, and Pasteurella, which are most commonly isolated from clinical samples of diseased animals in veterinary diagnostic laboratories, is presented in the current study. The identification procedure was evaluated with 40 type and reference strains and with 267 strains from routine diagnostic analysis of various animal species, including 28 different bacterial species. Type, reference, and field strains were analyzed by 16S ribosomal RNA (rrs) and rpoB gene sequencing for unambiguous species determination as a basis to evaluate the phenotypic differentiation schema. Primary phenotypic differentiation is based on beta-nicotinamide adenine dinucleotide (beta-NAD) dependence and hemolysis, which are readily determined on the isolation medium. The procedure divides the 28 species into 4 groups for which particular biochemical reactions were chosen to identify the bacterial species. The phenotypic identification procedure allowed researchers to determine the species of 240 out of 267 field strains. The procedure is an easy and cost-effective system for the rapid identification of species of the Pasteurellaceae family isolated from clinical specimens of animals.

Laribacter hongkongensis: a potential cause of infectious diarrhea

In this study, we describe the isolation of Laribacter hongkongensis, a recently described genus and species of bacterium, in pure culture on charcoal cefoperazone deoxycholate agar from the stool of six patients with diarrhea. Three patients were residents of Hong Kong, and three of Switzerland. In none of the stool samples obtained from these six patients was Salmonella, Shigella, enterohemorrhagic Escherichia coli, Vibrio, Aeromonas, Plesiomonas, or Campylobacter recovered. Rotavirus antigen detection, electron microscopic examination for viruses, and microscopic examinations for ova and cysts were all negative for the stool samples obtained from the three patients in Hong Kong. Enterotoxigenic E. coli was recovered from one of the patients in Hong Kong. Unlike L. hongkongensis type strain HKU1, all the six strains were motile with bipolar flagellae. Sequencing of the 16S ribosomal RNA genes of the six strains showed that they all had sequences with only 0-2 base differences to that of the type strain. Pulsed field gel electrophoresis of the SpeI digested genomic DNA of the six isolates and that of the type strain revealed that the seven isolates were genotypically unrelated strains. More extensive epidemiologic studies should be carried out to ascertain the causative association between L. hongkongensis and diarrhea and to define the reservoir and modes of transmission of L. hongkongensis.

Tularemia in a common marmoset (Callithrix jacchus) diagnosed by 16S rRNA sequencing

We report a case of tularemia in a common marmoset (Callithrix jacchus) diagnosed by determination of the isolate's 16S ribosomal RNA (rRNA) gene sequence. Pathological examination of the animal revealed a multifocal acute necrotizing hepatitis, interstitial nephritis, splenitis, and lymphangitis of the mandibular, retropharyngeal, and cervical and mesenteric lymph nodes. Moreover, multiple foci of acute necrosis were found in the epithelium of the jejunum and the interstitium of the lung. Bacteriological investigations revealed a septicemia. The isolated infectious agent was uncommon, not routinely diagnosed in our laboratory and therefore difficult to identify by conventional tools in a reasonable time and effort. thus, we decided to perform a genetic analysis based on the 16S rRNA gene sequence. Thereby, an infection with Francisella tularensis, the causative agent of tularemia, was unambiguously diagnosed. This shows the great advantage 16S rRNA gene sequencing has as a general identification approach for unusual or rare isolates.

Subgingival microbiota of Sri Lankan tea labourers naïve to oral hygiene measures.

AIM To characterize the subgingival microbiota within a cohort of adult males (n = 32) naïve to oral hygiene practices, and to compare the composition of bacterial taxa present in periodontal sites with various probing depths. MATERIAL AND METHODS Subgingival plaque samples were collected from single shallow pocket [pocket probing depth (PPD)≤3 mm] and deep pocket (PPD≥6 mm) sites from each subject. A polymerase chain reaction based strategy was used to construct a clone library of 16S ribosomal RNA (rRNA) genes for each site. The sequences of ca. 30-60 plasmid clones were determined for each site to identify resident taxa. Microbial composition was compared using a variety of statistical and bioinformatics approaches. RESULTS A total of 1887 cloned 16S rRNA gene sequences were analysed, which were assigned to 318 operational taxonomic units (98% identity cut-off). The subgingival microbiota was dominated by Firmicutes (69.8%), Proteobacteria (16.3%), and Fusobacteria (8.0%). The overall composition of microbial communities in shallow sites was significantly different from those within deep sites (∫-Libshuff, p < 0.001). CONCLUSIONS A taxonomically diverse subgingival microbiota was present within this cohort; however, the structures of the microbial communities present in the respective subjects exhibited limited variation. Deep and shallow sites contained notably different microbial compositions, but this was not correlated with the rate of periodontal progression.

Identification of animal Pasteurellaceae by MALDI-TOF mass spectrometry

Species of the family Pasteurellaceae play an important role as primary or opportunistic, predominantly respiratory, pathogens in domestic and wild animals. Some of them cause severe disease with high economic losses in commercial animal husbandry. Hence, rapid and accurate differentiation of Pasteurellaceae is important and signifies a particular challenge to diagnostic laboratories. Identification and differentiation of Pasteurellaceae is mostly done using phenotypic tests or genetic identification based on sequence similarity of housekeeping genes, such as the rrs gene encoding the 16S ribosomal RNA (16S rRNA). Both approaches are time consuming, laborious, and costly, therefore often delaying the final diagnosis of disease or epidemics. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry represents an alternative rapid and reliable method for the differentiation of most members of the family Pasteurellaceae. It is able to differentiate within a few minutes the currently known 18 genera and most of the over 60 species and subspecies of Pasteurellaceae including many members encountered in veterinary diagnostic laboratories. A few closely related species and subspecies that cannot be discriminated by MALDI-TOF are easily identified further by complementary simple tests, such as hemolysis done simultaneously or routinely during pathogen isolation.

Xpd/Ercc2 regulates CAK activity and mitotic progression

General transcription factor IIH (TFIIH) consists of nine sub- units: cyclin-dependent kinase 7 (Cdk7), cyclin H and MAT1 (forming the Cdk-activating-kinase or CAK complex), the two helicases Xpb/Hay and Xpd, and p34, p44, p52 and p62 (refs 1–3). As the kinase subunit of TFIIH, Cdk7 participates in basal transcription by phosphorylating the carboxy-terminal domain of the largest subunit of RNA polymerase II1,4,5. As part of CAK, Cdk7 also phosphorylates other Cdks, an essential step for their activation6–9. Here we show that the Drosophila TFIIH com- ponent Xpd negatively regulates the cell cycle function of Cdk7, the CAK activity. Excess Xpd titrates CAK activity, resulting in decreased Cdk T-loop phosphorylation, mitotic defects and lethality, whereas a decrease in Xpd results in increased CAK activity and cell proliferation. Moreover, Xpd is downregulated at the beginning of mitosis when Cdk1, a cell cycle target of Cdk7, is most active. Downregulation of Xpd thus seems to contribute to the upregulation of mitotic CAK activity and to regulate mitotic progression positively. Simultaneously, the downregulation of Xpd might be a major mechanism of mitotic silencing of basal transcription.

Mechanistic insights into tRNA translocation on the ribosome

The ribosome is a highly conserved cellular complex and constitutes the center of protein biosynthesis. As the ribosome consists to about 2/3 of ribosomal RNA (rRNA), the rRNA is involved in most steps of translation. In order to investigate the role of some defined rRNA residues in different aspects of translation we use the atomic mutagenesis approach. This method allows the site-specific incorporation of unnatural nucleosides into the rRNA in the context of the complete 70S from Thermus aquaticus, and thereby exceeds the possibilities of conventional mutagenesis. We first studied ribosome-stimulated EF-G GTP hydrolysis. Here, we could show that the non-bridging phosphate oxygen of A2662, which is part of the Sarcin-Ricin-Loop, is required for EF-G GTPase activation by the ribosome. EF-G GTPase is a crucial step for tRNA translocation from the A- to the P-site, and from the P- to the E-site, respectively. We furthermore used the atomic mutagenesis approach to more precisely characterize the 23S rRNA functional groups involved in E-site tRNA binding. While the ribosomal A- and P-sites have been functionally well characterized in the past, the contribution of the E-site to protein biosynthesis is still poorly understood in molecular terms. Our data disclose the importance of the highly conserved E-site base pair G2421-C2395 for effective translation. Ribosomes with a disrupted G2421-C2395 base pair are defective in tRNA binding to the E-site. This results in an impaired translation of genuine mRNAs, while homo-polymeric templates are not affected. Cumulatively our data emphasize the importance of E-site tRNA occupancy and in particular the intactness of the 23S rRNA base pair G2421-C2395 for productive protein biosynthesis.

DNA probes for the detection of Fasciola hepatica in snails

Fasciola hepatica, also called the large liver fluke, is a trematode which can infect most mammals. Monitoring the infection rate of snails, which function as intermediate hosts and harbour larval stages of F. hepatica, is an important component of epidemiological studies on fascioliasis. For this purpose, DNA probes were generated which can be used for the detection of F. hepatica larvae in snails. Four highly repetitive DNA fragments were cloned in a plasmid vector and tested by Southern blot hybridization to the DNA of various trematodes for specificity and sensitivity. The probes Fhr-I, Fhr-II and Fhr-III hybridized only to F. hepatica DNA. Fhr-IV contained ribosomal RNA gene sequences and cross-hybridize with the DNA from various other trematode species. Squash blot analysis showed that the different probes were able to detect the parasite larvae in trematode-infected snails even as isolated single larvae. No signals were obtained in squash blots of uninfected snails. Probes Fhr-I, Fhr-II and Fhr-III are thus useful specific tools for studying the epidemiology of fascioliasis. The probe Fhr-IV, because of its broader spectrum, can be used to detect the larvae of a wide range of trematode species of waterbirds, which are the causative agents of swimmer's itch.

tRNA-derived fragments target the small ribosomal subunit to fine-tune translation

Post-transcriptional cleavage of RNA molecules to generate smaller fragments is a widespread mechanism that enlarges the structural and functional complexity of cellular RNomes. In particular, fragments deriving from both precursor and mature tRNAs represent one of the rapidly growing classes of post-transcriptional RNA pieces. Importantly, these tRNA-derived fragments (tRFs) possess distinct expression patterns, abundance, cellular localizations, or biological roles compared with their parental tRNA molecules (1). Here we present evidence that tRFs from the archaeon Haloferax volcanii directly bind to ribosomes. In a previous genomic screen for ribosome-associated small RNAs we have identified a 26 residue long fragment originating from the 5’ part of valine tRNA (Val-tRF) to be by far the most abundant tRF in H. volcanii (2). The Val-tRF is processed in a stress- dependent manner and was found to primarily target the small ribosomal subunit in vitro and in vivo. Translational activity was markedly reduced in the presence of Val-tRF, while control RNA fragments of similar length did not show inhibition of protein biosynthesis. Crosslinking experiments and subsequent primer extension analyses revealed the Val-tRF interaction site to surround the mRNA path in the 30S subunit. In support of this, binding experiments demonstrated that Val-tRF does compete with mRNAs for ribosome binding. Therefore this tRF represents a ribosome-bound non-protein-coding RNA (ncRNA) capable of regulating gene expression in H. volcanii under environmental stress conditions probably by fine-tuning the rate of protein production (1). (1) Gebetsberger J. and Polacek N. (2013), RNA Biol. 10:1798-1808 (2) Gebetsberger J. et. al. (2012), Archaea, Article ID 260909

tRNA-derived fragments target the small ribosomal subunit to fine-tune translation

Post-transcriptional cleavage of RNA molecules to generate smaller fragments is a widespread mechanism that enlarges the structural and functional complexity of cellular RNomes. In particular, fragments deriving from both precursor and mature tRNAs represent one of the rapidly growing classes of post-transcriptional RNA pieces. Importantly, these tRNA-derived fragments (tRFs) possess distinct expression patterns, abundance, cellular localizations, or biological roles compared with their parental tRNA molecules [1]. Here we present evidence that tRFs from the archaeon Haloferax volcanii directly bind to ribosomes. In a previous genomic screen for ribosome-associated small RNAs we have identified a 26 residue long fragment originating from the 5’ part of valine tRNA (Val-tRF) to be by far the most abundant tRF in H. volcanii [2]. The Val-tRF is processed in a stress- dependent manner and was found to primarily target the small ribosomal subunit in vitro and in vivo. Translational activity was markedly reduced in the presence of Val-tRF, while control RNA fragments of similar length did not show inhibition of protein biosynthesis. Crosslinking experiments and subsequent primer extension analyses revealed the Val-tRF interaction site to surround the mRNA path in the 30S subunit. In support of this, binding experiments demonstrated that Val-tRF does compete with mRNAs for ribosome binding. Therefore this tRF represents a ribosome-bound non-protein-coding RNA (ncRNA) capable of regulating gene expression in H. volcanii under environmental stress conditions probably by fine-tuning the rate of protein production [1].

tRNA-derived fragments target the small ribosomal subunit to fine-tune translation

Post-transcriptional cleavage of RNA molecules to generate smaller fragments is a widespread mechanism that enlarges the structural and functional complexity of cellular RNomes. In particular, fragments deriving from both precursor and mature tRNAs represent one of the rapidly growing classes of post-transcriptional RNA pieces. Importantly, these tRNA-derived fragments (tRFs) possess distinct expression patterns, abundance, cellular localizations, or biological roles compared with their parental tRNA molecules [1]. Here we present evidence that tRFs from the archaeon Haloferax volcanii directly bind to ribosomes. In a previous genomic screen for ribosome-associated small RNAs we have identified a 26 residue long fragment originating from the 5’ part of valine tRNA (Val-tRF) to be by far the most abundant tRF in H. volcanii [2]. The Val-tRF is processed in a stress- dependent manner and was found to primarily target the small ribosomal subunit in vitro and in vivo. Translational activity was markedly reduced in the presence of Val-tRF, while control RNA fragments of similar length did not show inhibition of protein biosynthesis. Crosslinking experiments and subsequent primer extension analyses revealed the Val-tRF interaction site to surround the mRNA path in the 30S subunit. In support of this, binding experiments demonstrated that Val-tRF does compete with mRNAs for ribosome binding. Therefore this tRF represents a ribosome-associated non-protein-coding RNA (rancRNA) capable of regulating gene expression in H. volcanii under environmental stress conditions probably by fine-tuning the rate of protein production [3].

tRNA-derived fragments target the small ribosomal subunit to fine-tune translation

Post-transcriptional cleavage of RNA molecules to generate smaller fragments is a widespread mechanism that enlarges the structural and functional complexity of cellular RNomes. In particular, fragments deriving from both precursor and mature tRNAs represent one of the rapidly growing classes of post-transcriptional RNA pieces. Importantly, these tRNA-derived fragments (tRFs) possess distinct expression patterns, abundance, cellular localizations, or biological roles compared with their parental tRNA molecules [1]. Here we present evidence that tRFs from the halophilic archaeon Haloferax volcanii directly bind to ribosomes. In a previous genomic screen for ribosome-associated small RNAs we have identified a 26 residue long fragment originating from the 5’ part of valine tRNA (Val-tRF) to be by far the most abundant tRF in H. volcanii [2]. The Val-tRF is processed in a stress-dependent manner and was found to primarily target the small ribosomal subunit in vitro and in vivo. Translational activity was markedly reduced in the presence of Val-tRF, while control RNA fragments of similar length did not show inhibition of protein biosynthesis. Crosslinking experiments and subsequent primer extension analyses revealed the Val-tRF interaction site to surround the mRNA path in the 30S subunit. In support of this, binding experiments demonstrated that Val-tRF does compete with mRNAs for ribosome binding. Therefore this tRF represents a ribosome-associated non-coding RNA (rancRNA) capable of regulating gene expression in H. volcanii under environmental stress conditions probably by fine-tuning the rate of protein production [3].

Crystal structure of the yeast eIF4A-eIF4G complex: an RNA-helicase controlled by protein-protein interactions

Translation initiation factors eIF4A and eIF4G form, together with the cap-binding factor eIF4E, the eIF4F complex, which is crucial for recruiting the small ribosomal subunit to the mRNA 5' end and for subsequent scanning and searching for the start codon. eIF4A is an ATP-dependent RNA helicase whose activity is stimulated by binding to eIF4G. We report here the structure of the complex formed by yeast eIF4G's middle domain and full-length eIF4A at 2.6-A resolution. eIF4A shows an extended conformation where eIF4G holds its crucial DEAD-box sequence motifs in a productive conformation, thus explaining the stimulation of eIF4A's activity. A hitherto undescribed interaction involves the amino acid Trp-579 of eIF4G. Mutation to alanine results in decreased binding to eIF4A and a temperature-sensitive phenotype of yeast cells that carry a Trp579Ala mutation as its sole source for eIF4G. Conformational changes between eIF4A's closed and open state provide a model for its RNA-helicase activity.

An intergenic non-coding RNA targets and regulates the ribosome in H. volcanii

As translation is the final step in gene expression it is particularly important to understand the processes involved in translation regulation. It was shown in the last years that a class of RNA, the non-protein-coding RNAs (ncRNAs), is involved in regulation of gene expression via various mechanisms (e.g. gene silencing by microRNAs). Almost all of these ncRNA discovered so far target the mRNA in order to modulate protein biosynthesis, this is rather unexpected considering the crucial role of the ribosome during gene expression. However, recent data from our laboratory showed that there is a new class of ncRNAs, which target the ribosome itself [Gebetsberger et al., 2012/ Pircher et al, 2014]. These so called ribosome-associated ncRNAs (rancRNAs) have an impact on translation regulation, mainly by interfering / modulating the rate of protein biosynthesis. The main goal of this project is to identify and describe novel potential regulatory rancRNAs in H. volcanii with the focus on intergenic candidates. Northern blot analyses already revealed interactions with the ribosome and showed differential expression of rancRNAs during different growth phases or under specific stress conditions. To investigate the biological relevance of these rancRNAs, knock-outs were generated in H. volcanii which were used for phenotypic characterization studies. The rancRNA s194 showed association with the 50S ribosomal subunit in vitro and in vivo and was capable of inhibiting peptide bond formation and seems to inhibit translation in vitro. These preliminary data for the rancRNA s194 make it an interesting candidate for further functional studies to identify the molecular mechanisms by which rancRNAs can modulate protein biosynthesis. Characterization of further rancRNA candidates are also underway.

An intergenic non-coding RNA targets and regulates the ribosome in H. volcanii.

As translation is the final step in gene expression it is particularly important to understand the processes involved in translation regulation. It was shown in the last years that a class of RNA, the nonprotein-coding RNAs (ncRNAs), is involved in regulation of gene expression via various mechanisms (e.g. gene silencing by microRNAs). Almost all of these ncRNA discovered so far target the mRNA in order to modulate protein biosynthesis, this is rather unexpected considering the crucial role of the ribosome during gene expression. However, recent data from our laboratory showed that there is a new class of ncRNAs, which target the ribosome itself [Gebetsberger et al., 2012/ Pircher et al, 2014]. These so called ribosome-associated ncRNAs (rancRNAs) have an impact on translation regulation, mainly by interfering / modulating the rate of protein biosynthesis. The main goal of this project is to identify and describe novel potential regulatory rancRNAs in H. volcanii with the focus on intergenic candidates. Northern blot analyses already revealed interactions with the ribosome and showed differential expression of rancRNAs during different growth phases or under specific stress conditions. To investigate the biological relevance of these rancRNAs, knock-outs were generated in H. volcanii which were used for phenotypic characterization studies. The rancRNA s194 showed association with the 50S ribosomal subunit in vitro and in vivo and was capable of inhibiting peptide bond formation. These preliminary data for the rancRNA s194 make it an interesting candidate for further functional studies to identify the molecular mechanisms by which rancRNAs can modulate protein biosynthesis. Characterization of further rancRNA candidates are also underway.