Pay32p of the Yeast Yarrowia lipolytica Is an Intraperoxisomal Component of the Matrix Protein Translocation Machinery

Pay mutants of the yeast Yarrowia lipolytica fail to assemble functional peroxisomes. One mutant strain, pay32-1, has abnormally small peroxisomes that are often found in clusters surrounded by membranous material. The functionally complementing gene PAY32 encodes a protein, Pay32p, of 598 amino acids (66,733 D) that is a member of the tetratricopeptide repeat family. Pay32p is intraperoxisomal. In wild-type peroxisomes, Pay32p is associated primarily with the inner surface of the peroxisomal membrane, but ~30% of Pay32p is localized to the peroxisomal matrix. The majority of Pay32p in the matrix is complexed with two polypeptides of 62 and 64 kD recognized by antibodies to SKL (peroxisomal targeting signal-1). In contrast, in peroxisomes of the pay32-1 mutant, Pay32p is localized exclusively to the matrix and forms no complex. Biochemical characterization of the mutants pay32-1 and pay32-KO (a PAY32 gene disruption strain) showed that Pay32p is a component of the peroxisomal translocation machinery. Mutations in the PAY32 gene prevent the translocation of most peroxisome-bound proteins into the peroxisomal matrix. These proteins, including the 62-kD anti-SKL-reactive polypeptide, are trapped in the peroxisomal membrane at an intermediate stage of translocation in pay32 mutants. Our results suggest that there are at least two distinct translocation machineries involved in the import of proteins into peroxisomes.

Novel methylotrophic bacteria isolated from the River Thames (London, UK)

Enrichment and elective culture for methylotrophs from sediment of the River Thames in central London yielded a diversity of pure cultures representing several genera of Gram-negative and Gram-positive bacteria, which were mainly of organisms not generally regarded as typically methylotrophic. Substrates leading to successful isolations included methanol, monomethylamine, dimethylamine, trimethylamine, methanesulfonate and dimethylsulfone. Several isolates were studied in detail and shown by their biochemical and morphological properties and 16S rRNA gene sequencing to be Sphingomonas melonis strain ET35, Mycobacterium fluoranthenivorans strain DSQ3, Rhodococcus erythropolis strain DSQ4, Brevibacterium casei strain MSQ5, Klebsiella oxytoca strains MMA/F and MMA/1, Pseudomonas mendocina strain TSQ4, and Flavobacterium sp. strains MSA/1 and MMA/2. The results show that facultative methylotrophy is present across a wide range of Bacteria, suggesting that turnover of diverse C1-compounds is of much greater microbiological and environmental significance than is generally thought. The origins of the genes encoding the enzymes of methylotrophy in diverse heterotrophs need further study, and could further our understanding of the phylogeny and antiquity of methylotrophic systems

Prevalence and diversity of Arcobacter spp. in poultry meat in New Zealand : a thesis presented in the partial fulfillment of the requirements for the degree of Master of Science in Veterinary Microbiology and Public Health at Massey University, Palmerston North, New Zealand

The microaerophilic bacterium Arcobacter has received increased attention in recent years as an emerging foodborne human pathogen. Although phenotypically related, arcobacters differ from campylobacters in their ability to grow aerobically and at lower temperatures. Poultry are considered a significant reservoir of this organism, with an isolation rate of up to 72% in faecal samples, and up to 100% in meat samples. To date, four species; A. butzleri, A. skirrowii, A. cryaerophilus, and A. cibarius have been isolated from poultry. The first three species have also been found to be associated with human and animal illnesses such as diarrhoea, bacteraemia, mastitis and abortions. The organisms are also found in raw meat products as well as in surface and ground water. Since most laboratories still do not use appropriate isolation techniques, the occurrence of this organism in food sources and their role in human illnesses is greatly underestimated. This is the first investigation of the prevalence of arcobacters in poultry meat in New Zealand. The aim of this study was to compare the most commonly used Arcobacter isolation methods. In addition, this study aimed to estimate the prevalence of Arcobacter spp. in retail poultry in New Zealand. Other aims include comparison of genetic diversity of Arcobacter spp. isolated from three different poultry producers, and by different methods, and estimation of overall genetic diversity of arcobacters present in New Zealand. During the period of May to October 2005, a total of 150 fresh, whole, retail poultry carcass produced by three different producers were purchased through two supermarket outlets in Palmerston North, New Zealand. Isolation of Arcobacter was done by seven different techniques. Arcobacter-like organisms were identified presumptively by phenotypic tests; temperature tolerance, aerotolerance, motility , and oxidase production. These presumptive arcobacters were confirmed by a species-specific multiplex PCR (m-PCR) either as A. butzleri, A. cryaerophilus or A. skirrowii. DNA sequencing was done for selected isolates from both species to further confirm the PCR results. The PCR positive isolates were subjected to Pulsed-Field Gel Electrophoresis (PFGE) following restriction digestion with Eagl. It was found that 55.3 % of 150 retail poultry sold in New Zealand were harbouring Arcobacter species. Two species; A. butzleri and A. cryaerophilus were detected by m-PCR which was later confirmed by sequencing. A total of 189 isolates were detected by six methods from 83 retail poultry samples. A. butzleri was the predominant species and was detected in 51.3% of the samples, whereas A. cryaerophilus was detected only in 8% of the samples. A. butzleri and A. cryaerophilus accounted for 92.6% (n=175) and 7.4% (n-14) of the isolates, respectively. A. butzleri was the only Arcobacter species present in 46.6% samples, and A. cryaerophilus only in 3.3% of the samples. Both species were detected simultaneously in 4.6% of the samples. There was a wide variation among the prevalence rate of Arcobacter spp. in retail poultry from different producers varying from 30 to 98%. There was also a wide variation among the isolation rates of different methods varying from 3.3 to 39.3%. The best isolation method was found to be Arcobacter-broth enrichment followed by passive filtration through a sterile filter of 0.45μm, onto blood-agar plates. No single isolation method detected all arcobacters. PFGE of Arcobacter isolates demonstrated the occurrence of multiple genotypes of both A. butzleri and A. cryaerophilus in the retail poultry from the same producers, and even in a single poultry. The possible explanations for the large amount of heterogeneity include multiple sources of contamination, the occurrence of multiple parent genotypes for both species in a single poultry carcass, and a high degree of genomic recombination among the progeny of historical parent genotypes. This study highlights the high prevalence of Arcobacter spp. in poultry meat in New Zealand. It also indicates prevalence of arcobacters in poultry carcass varies greatly with the choice of isolation method and none of the currently available methods are appropriate for the detection of all species of arcobacters in New Zealand. Therefore, two or more methods should be used in parallel. The level of contamination of poultry carcass may vary with the processing practices of a slaughterhouse. To eliminate or reduce arcobacters in retail poultry, maintenance of slaughter hygiene is of utmost importance. This may be achieved by regular microbiological monitoring of carcasses according to the HACCP principles. Further studies comparing the fingerprinting pattern of Arcobacter spp. isolates obtained from retails poultry with human isolates are necessary to test the hypothesis that poultry meal is an important source for Arcobacter infection in human

Characterization of Francisella species isolated from the cooling water of an air conditioning system.

Strains of Francisella spp. were isolated from cooling water from an air conditioning system in Guangzhou, China. These strains are Gram negative, coccobacilli, non-motile, oxidase negative, catalase negative, esterase and lipid esterase positive. In addition, these bacteria grow on cysteine-supplemented media at 20 °C to 40 °C with an optimal growth temperature of 30 °C. Analysis of 16S rRNA gene sequences revealed that these strains belong to the genus Francisella. Biochemical tests and phylogenetic and BLAST analyses of 16S rRNA, rpoB and sdhA genes indicated that one strain was very similar to Francisella philomiragia and that the other strains were identical or highly similar to the Francisella guangzhouensis sp. nov. strain 08HL01032 we previously described. Biochemical and molecular characteristics of these strains demonstrated that multiple Francisella species exist in air conditioning systems

A comprehensive evaluation of colonic mucosal isolates of Sutterella wadsworthensis from inflammatory bowel disease

Peer reviewedPublisher PD

Potentially Diagnostic Electron Paramagnetic Resonance Spectra Elucidate the Underlying Mechanism of Mitochondrial Dysfunction in the Deoxyguanosine Kinase Deficient Rat Model of a Genetic Mitochondrial DNA Depletion Syndrome

A novel rat model for a well-characterized human mitochondrial disease, mitochondrial DNA depletion syndrome with associated deoxyguanosine kinase (DGUOK) deficiency, is described. The rat model recapitulates the pathologic and biochemical signatures of the human disease. The application of electron paramagnetic (spin) resonance (EPR) spectroscopy to the identification and characterization of respiratory chain abnormalities in the mitochondria from freshly frozen tissue of the mitochondrial disease model rat is introduced. EPR is shown to be a sensitive technique for detecting mitochondrial functional abnormalities in situ and, here, is particularly useful in characterizing the redox state changes and oxidative stress that can result from depressed expression and/or diminished specific activity of the distinct respiratory chain complexes. As EPR requires no sample preparation or non-physiological reagents, it provides information on the status of the mitochondrion as it was in the functioning state. On its own, this information is of use in identifying respiratory chain dysfunction; in conjunction with other techniques, the information from EPR shows how the respiratory chain is affected at the molecular level by the dysfunction. It is proposed that EPR has a role in mechanistic pathophysiological studies of mitochondrial disease and could be used to study the impact of new treatment modalities or as an additional diagnostic tool

The Hansenula polymorpha PER8 Gene Encodes a Novel Peroxisomal Integral Membrane Protein Involved in Proliferation

We previously described the isolation of mutants of the methylotrophic yeast Hansenula polymorpha that are defective in peroxisome biogenesis. Here, we describe the characterization of one of these mutants, per8, and the cloning of the PER8 gene. In either methanol or methylamine medium, conditions that normally induce the organdies, per8 cells contain no peroxisome-like structures and peroxisomal enzymes are located in the cytosol. The sequence of PER8 predicts that its product (Per8p) is a novel polypeptide of 34 kD, and antibodies against Per8p recognize a protein of 31 kD. Analysis of the primary sequence of Per8p revealed a 39-amino-acid cysteine-rich segment with similarity to the C3HC4 family of zinc-finger motifs. Overexpression of PER8 results in a markedly enhanced increase in peroxisome numbers. We show that Per8p is an integral membrane protein of the peroxisome and that it is concentrated in the membranes of newly formed organdies. We propose that Per8p is a component of the molecular machinery that controls the proliferation of this organelle.

Description of Treponema azotonutricium sp. nov. and Treponema primitia sp. nov., the First Spirochetes Isolated from Termite Guts

Long after their original discovery, termite gut spirochetes were recently isolated in pure culture for the first time. They revealed metabolic capabilities hitherto unknown in the Spirochaetes division of the Bacteria, i.e., H2 plus CO2 acetogenesis (J. R. Leadbetter, T. M. Schmidt, J. R. Graber, and J. A. Breznak, Science 283:686-689, 1999) and dinitrogen fixation (T. G. Lilburn, K. S. Kim, N. E. Ostrom, K. R. Byzek, J. R. Leadbetter, and J. A. Breznak, Science 292:2495-2498, 2001). However, application of specific epithets to the strains isolated (Treponema strains ZAS-1, ZAS-2, and ZAS-9) was postponed pending a more complete characterization of their phenotypic properties. Here we describe the major properties of strain ZAS-9, which is readily distinguished from strains ZAS-1 and ZAS-2 by its shorter mean cell wavelength or body pitch (1.1 versus 2.3 µm), by its nonhomoacetogenic fermentation of carbohydrates to acetate, ethanol, H2, and CO2, and by 7 to 8% dissimilarity between its 16S rRNA sequence and those of ZAS-1 and ZAS-2. Strain ZAS-9 is proposed as the type strain of the new species, Treponema azotonutricium. Strains ZAS-1 and ZAS-2, which are H2-consuming, CO2-reducing homoacetogens, are proposed here to be two strains of the new species Treponema primitia. Apart from the salient differences mentioned above, the genomes of all three strains were similar in size (3,461 to 3,901 kb), in G+C content (50.0 to 51.0 mol%), and in possession of 2 copies of the gene encoding 16S rRNA (rrs). For comparison, the genome of the free-living spirochete Spirochaeta aurantia strain J1 was analyzed by the same methods and found to have a size of 3,719 kb, to contain 65.6 mol% G+C, and also to possess 2 copies of the rrs gene

Phylogenomic study of Burkholderia glathei-like organisms, proposal of 13 novel Burkholderia species and emended descriptions of Burkholderia sordidicola, Burkholderia zhejiangensis, and Burkholderia grimmiae

Partial gyrB gene sequence analysis of 17 isolates from human and environmental sources revealed 13 clusters of strains and identified them as Burkholderia glathei Glade (BGC) bacteria. The taxonomic status of these clusters was examined by whole-genome sequence analysis, determination of the G+C content, whole-cell fatty acid analysis and biochemical characterization. The whole-genome sequence-based phylogeny was assessed using the Genome Blast Distance Phylogeny (GBDP) method and an extended multilocus sequence analysis (MLSA) approach. The results demonstrated that these 17 BGC isolates represented 13 novel Burkholderia species that could be distinguished by both genotypic and phenotypic characteristics. BGC strains exhibited a broad metabolic versatility and developed beneficial, symbiotic, and pathogenic interactions with different hosts. Our data also confirmed that there is no phylogenetic subdivision in the genus Burkholderia that distinguishes beneficial from pathogenic strains. We therefore propose to formally classify the 13 novel BGC Burkholderia species as Burkholderia arvi sp. nov. (type strain LMG 29317(T) = CCUG 68412(T)), Burkholderia hypogeia sp. nov. (type strain LMG 29322(T) = CCUG 68407(T)), Burkholderia ptereochthonis sp. nov. (type strain LMG 29326(T) = CCUG 68403(T)), Burkholderia glebae sp. nov. (type strain LMG 29325(T) = CCUG 68404(T)), Burkholderia pedi sp. nov. (type strain LMG 29323(T) = CCUG 68406(T)), Burkholderia arationis sp. nov. (type strain LMG 29324(T) = CCUG 68405(T)), Burkholderia fortuita sp. nov. (type strain LMG 29320(T) = CCUG 68409(T)), Burkholderia temeraria sp. nov. (type strain LMG 29319(T) = CCUG 68410(T)), Burkholderia calidae sp. nov. (type strain LMG 29321(T) = CCUG 68408(T)), Burkholderia concitans sp. nov. (type strain LMG 29315(T) = CCUG 68414(T)), Burkholderia turbans sp. nov. (type strain LMG 29316(T) = CCUG 68413(T)), Burkholderia catudaia sp. nov. (type strain LMG 29318(T) = CCUG 68411(T)) and Burkholderia peredens sp. nov. (type strain LMG 29314(T) = CCUG 68415(T)). Furthermore, we present emended descriptions of the species Burkholderia sordidicola, Burkholderia zhejlangensis and Burkholderia grimmiae. The GenBank/EMBUDDBJ accession numbers for the 16S rRNA and gyrB gene sequences determined in this study are LT158612-LT158624 and LT158625-LT1158641, respectively

Diversity of Xenorhabdus and Photorhabdus spp. and their symbiotic entomopathogenic nematodes from Thailand

Xenorhabdus and Photorhabdus spp. are bacterial symbionts of entomopathogenic nematodes (EPNs). In this study, we isolated and characterized Xenorhabdus and Photorhabdus spp. from across Thailand together with their associated nematode symbionts, and characterized their phylogenetic diversity. EPNs were isolated from soil samples using a Galleria-baiting technique. Bacteria from EPNs were cultured and genotyped based on recA sequence. The nematodes were identified based on sequences of 28S rDNA and internal transcribed spacer regions. A total of 795 soil samples were collected from 159 sites in 13 provinces across Thailand. A total of 126 EPNs isolated from samples taken from 10 provinces were positive for Xenorhabdus (n = 69) or Photorhabdus spp. (n = 57). Phylogenetic analysis separated the 69 Xenorhabdus isolates into 4 groups. Groups 1, 2 and 3 consisting of 52, 13 and 1 isolates related to X. stockiae, and group 4 consisting of 3 isolates related to X. miraniensis. The EPN host for isolates related to X. stockiae was S. websteri, and for X. miraniensis was S. khoisanae. The Photorhabdus species were identified as P. luminescens (n = 56) and P. asymbiotica (n = 1). Phylogenenic analysis divided P. luminescens into five groups. Groups 1 and 2 consisted of 45 and 8 isolates defined as subspecies hainanensis and akhurstii, respectively. One isolate was related to hainanensis and akhurstii, two isolates were related to laumondii, and one isolate was the pathogenic species P. asymbiotica subsp. australis. H. indica was the major EPN host for Photorhabdus. This study reveals the genetic diversity of Xenorhabdus and Photorhabdus spp. and describes new associations between EPNs and their bacterial symbionts in Thailand