RESUMEN
The 16S-23S rDNA internal transcribed spacer (ITS) polymorphism analysis was assessed for its suitability in rapid discrimination between species of psychrophilic and psychrotolerant clostridia associated with "blown pack" spoilage of vacuum-packed meats. DNA isolated from 10 reference and 20 meat strains of psychrophilic and psychrotolerant clostridia were used as templates in PCR amplification with primers complementary to conserved regions of the 3' end of the 16S rRNA and 5' end of the 23S rRNA genes directly flanking the spacer. The majority of strains showed multi-band ITS patterns when products of spacer amplification were visualised on an agarose gel. With the majority of meat strains, PCR amplification generated single banding pattern for a single clostridial species. However, meat strains of Cl. algidicarnis produced four different ITS banding patterns. With reference strains of psychrophilic and psychrotolerant clostridia, variation in spacer length was also observed between nonproteolytic Cl. botulinum type B (17B), E (Beluga) and F (202F). On the other hand, the number and size of the ITS amplification products could not be used for a differentiation of Cl. laramiense ATCC 51254(T) from Cl. estertheticum DSM 8809(T), Cl. putrefaciens DSM 1291(T) from Cl. algidicarnis NCFB 2931(T), or Cl. frigidicarnis strains from nonproteolytic Cl. botulinum type B (17B). The presence of interstrain, and lack of interspecies, ITS polymorphism observed in the present study with some clostridial species may preclude the use of 16S-23S rDNA spacer amplification for species-level discrimination and identification, respectively, of psychrophilic and psychrotolerant clostridia associated with meat spoilage. However, where interstrain, intraspecies heterogeneity of ITS amplification products exists, ITS analysis could be useful for tracing back psychrophilic and psychrotolerant clostridia responsible for meat spoilage to their meat plant sources.
Asunto(s)
Clostridium/clasificación , Microbiología de Alimentos , Embalaje de Alimentos/métodos , Carne/microbiología , Animales , Clostridium/genética , ADN Bacteriano/análisis , ADN Ribosómico/análisis , ADN Espaciador Ribosómico/análisis , Amplificación de Genes , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Análisis de Secuencia de ADN , Especificidad de la Especie , VacioRESUMEN
AIMS: To identify the abattoir source(s) of culturable psychrophilic clostridia causing 'blown pack' spoilage of vacuum-packed chilled meats. METHODS AND RESULTS: Psychrophilic and psychrotolerant clostridia were isolated from hides, faeces and tonsils of deer slaughter stock, and from a meat plant environment. The isolates were differentiated using restriction fragment length polymorphism analysis of the 16S rDNA gene (PCR-RFLP) and 16S-23S rDNA internal transcribed spacer (ITS) analysis. PCR-RFLP group I clostridia were found to have restriction patterns indistinguishable from the patterns of 'blown pack'-causing Clostridium gasigenes DB1A(T) and R26. Gas production in packs inoculated with vegetative cells of PCR-RFLP group I clostridia was first evident after 14 days at 2 degrees C. The prevalence of these clostridia was similar in hide and faecal samples from slaughter animals, but these micro-organisms were absent from tonsils and the meat plant environment. Banding patterns of PCR-RFLP group II clostridia showed some cross-similarity with patterns of the 'blown pack'-causing micro-organism Cl. estertheticum DSM 8809(T) and Cl. estertheticum-like meat strains. The majority of clostridia in PCR-RFLP group II were found in the faeces of slaughter animals. Isolates representing PCR-RFLP group II did not, however, produce gas in vacuum packs stored at 2 degrees C for 84 days. CONCLUSIONS: The data suggest that soil particles attached to hide or present in faeces are the most probable primary reservoir from which 'blown pack' clostridia are introduced onto carcasses. Therefore, dressing procedure hygiene remains paramount in order to control the spread of psychrophilic Clostridium spp. in a meat plant. SIGNIFICANCE AND IMPACT OF THE STUDY: The paper provides information critical for controlling 'blown pack' spoilage in meat processing plants. It reports on the use of molecular techniques for determination of abattoir sources of 'blown pack'-causing clostridia.
Asunto(s)
Mataderos , Clostridium/aislamiento & purificación , Frío , Ciervos , Microbiología de Alimentos , Productos de la Carne/microbiología , Animales , Clostridium/genética , Clostridium/crecimiento & desarrollo , ADN Bacteriano/análisis , ADN Ribosómico/análisis , ADN Espaciador Ribosómico/química , ADN Espaciador Ribosómico/genética , ADN Espaciador Ribosómico/aislamiento & purificación , Heces/microbiología , Manipulación de Alimentos , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , ARN Ribosómico 16S/análisis , Piel/microbiología , VacioRESUMEN
HTz is a member of the archaeal histone family. The archaeal histones have primary sequences and structural similarity to the eukaryal histone fold domain, and are thought to resemble the archetypal ancestor of the eukaryal nucleosome core histones. The effects of growth phase on the total soluble proteins from Thermococcus zilligii, isolated after various stages of growth from mid-logarithmic to late stationary phase, were examined by denaturing polyacrylamide gel electrophoresis. On entry into stationary phase, at least 11 proteins were detected that changed considerably in level. One of these proteins was identified by Western hybridization as HTz. The level of HTz decreased dramatically as cells entered stationary phase, and it could not be detected by late stationary phase. Unexpectedly, the Western hybridization detected a second protein, with an estimated molecular mass of approximately 14 kDa, which paralleled the decrease in level of HTz. Native purified HTz was shown to retain complete activity after prolonged incubation at the growth temperature of the organism, suggesting that the decrease in HTz was a specific cell-regulated process. Analysis of native purified HTz by electrospray ionization mass spectrometry revealed the molecular masses of HTz1 and HTz2 to be 7204 +/- 3 Da and 7016 +/- 3 Da respectively. The only non-covalent species that was detected corresponded to the molecular mass of an HTz1-HTz2 heterodimer. Northern analyses of T. zilligii total RNA with an htz1 gene probe indicated a rapid decrease in expression of htz1 with progression of the growth phase, and complete repression of htz1 transcript synthesis by late logarithmic phase. Three proteins that changed in level with growth phase were identified by N-terminal sequence analysis. The first was homologous to a hypothetical protein conserved in all Archaea sequenced to date, the second to the Sac10b family of archaeal DNA-binding proteins and the third to the C-terminal region of the leucine-responsive regulatory family of DNA-binding proteins (LRPs).
Asunto(s)
Proteínas Arqueales/metabolismo , Histonas/metabolismo , Thermococcus/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Arqueales/genética , Dimerización , Expresión Génica , Histonas/genética , Datos de Secuencia Molecular , Conejos , Soluciones , Temperatura , Thermococcus/genética , Thermococcus/crecimiento & desarrollo , Transcripción GenéticaRESUMEN
Two psychrophilic Clostridium strains, DB1AT and R26, were isolated from incidences of 'blown-pack' spoilage of vacuum-packed chilled lamb. Vacuum packs of meat inoculated with these strains developed gas bubbles and pack distension within 14 d storage at 2 degrees C. The two main gases responsible for pack distension were carbon dioxide and hydrogen. 1-Butanol, butyric and acetic acid and butyl esters were the major volatile compounds produced by the strains in the artificially inoculated packs. The unknown strains were Gram-positive motile rods producing elliptical subterminal spores during the late-stationary growth phase. At pH 7.0, they grew from -1.5 to 26 degrees C, and their optimum growth temperature was 20-22 degrees C. At 20 degrees C, the pH range for growth was 5.4-8.9 and the optimum pH for growth was 6.2-8.6. In peptone/yeast extract broth, the organisms grew little or not at all in the absence of fermentable carbohydrates. Both strains hydrolysed gelatin, aesculin and starch. The fermentation products formed in peptone yeast extract glucose starch broth were ethanol, acetate, butyrate, lactate, butanol, carbon dioxide and hydrogen. The G+C contents of the DNA of strains DB1AT and R26 were 29.4 and 28.3 mol%, respectively. Phylogenetic analyses indicated that the strains belong to cluster I of the genus Clostridium (sensu Collins et al. 1994). The new strains differed from the phylogenetically related clostridia in cellular fatty acid composition, soluble protein profiles and phenotypic properties. On the basis of rDNA analysis and phenotypic and phylogenetic characterization, the strains were assigned to a new species for which the name Clostridium gasigenes is proposed. Strain DB1AT (= DSM 12272T) is designated as the type strain.
Asunto(s)
Clostridium/clasificación , Frío , Manipulación de Alimentos , Embalaje de Alimentos , Carne/microbiología , Composición de Base , Butanoles/metabolismo , Ácido Butírico/metabolismo , Clostridium/aislamiento & purificación , Clostridium/fisiología , Clostridium/ultraestructura , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Fermentación , Gases , Genes de ARNr/genética , Datos de Secuencia Molecular , Fenotipo , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , VacioRESUMEN
A novel stationary phase-response protein has been identified in the acid-soluble protein extract of the thermophilic archaeon, Thermococcus zilligii. N-Terminal sequencing data were used to identify likely genes for homologues of the protein in the complete genome sequences of various archaeal species. The corresponding genes were identified and analysed. The genes encode a protein ranging from 83 to 92 amino acids in length, with a calculated pI ranging from 4.6 to 9.7. The amino acid sequences of the genes were highly conserved, even between members belonging to the different archaeal kingdoms. The computed secondary structure of the protein indicates it consists of four large helical regions separated by short coiled regions. We propose this protein as a candidate regulator of gene expression in stationary phase.
Asunto(s)
Archaea/genética , Proteínas Arqueales/genética , Secuencia de Aminoácidos , Archaea/fisiología , Proteínas Arqueales/química , Proteínas Arqueales/aislamiento & purificación , Secuencia de Bases , Histonas/química , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Alineación de Secuencia , Thermococcus/genéticaRESUMEN
A psychrotolerant Clostridium species was isolated from vacuum-packed, temperature-abused raw lamb. Colonies of this micro-organism on sheep-blood agar were circular with an entire margin, grey-white, translucent and beta-haemolytic. Cells were single, tapered, motile rods. Elliptical subterminal spores were produced in the late stationary growth phase. Spores did not cause swelling of the maternal cells. The micro-organism was obligately anaerobic. In peptone yeast extract glucose starch (PYGS) broth at pH 7.0, the micro-organism grew optimally between 25.5 and 30.0 degrees C. The temperature range for growth was 2.5-32.2 degrees C. At 26 degrees C, the micro-organism grew optimally at pH 6.8 to 7.0. The pH range for anaerobic growth was 4.7-9.1. The micro-organism was saccharoclastic, hydrolysed starch and degraded xylan. The fermentation products formed in PYGS broth were acetate, formate, lactate, ethanol, butyrate, butanol, hydrogen and carbon dioxide. The G + C content of the DNA was 38.4 mol%. Phylogenetic analyses indicated that the strain belongs to cluster XIVa of the genus Clostridium (sensu Collins et al. 1994). The new strain differed from phylogenetically related clostridia in terms of cellular fatty acid composition, soluble protein profiles and phenotypic properties. On the basis of phenotypic and genotypic characterization data, the strain was assigned to a new species, namely Clostridium algidixylanolyticum. The type strain is strain SPL73T (= DSM 12273T).
Asunto(s)
Clostridium/clasificación , Frío , Embalaje de Alimentos , Carne/microbiología , Xilanos/metabolismo , Animales , Composición de Base , Clostridium/metabolismo , Clostridium/fisiología , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Conservación de Alimentos , Concentración de Iones de Hidrógeno , Ratones , Datos de Secuencia Molecular , Fenotipo , Filogenia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Ovinos , Esporas Bacterianas/fisiología , VacioRESUMEN
Reference and meat strains of psychrophilic and psychrotrophic clostridia were differentiated using restriction fragment length polymorphism (RFLP) analysis of genomic DNA (DNA-RFLP) and the polymerase chain reaction-amplified 16S rDNA gene (PCR-RFLP). Groupings obtained with PCR-RFLP were confirmed with 16S rDNA gene sequencing. DNA-RFLP resolved 19 of the 22 meat strains into 11 groups. Three meat strains were untypable using this method. All reference strains representing different genotypic species could be distinguished by the restriction patterns of 16S rDNA genes. With PCR-RFLP, the 22 meat strains produced eight distinct genotypes. 16S rDNA gene sequencing confirmed that each genotype was represented by a distinct sequence. PCR-RFLP restriction patterns of 15 meat strains matched those of one of two of the seven reference strains used. Seven meat strains whose RFLP restriction patterns of 16S rDNA genes differed from those of any reference strains probably represent four previously undescribed species. Although RFLP analysis of the amplified 16S rDNA gene allowed differentiation of psychrophilic and psychrotrophic clostridia at the genotypic species level and below, comparison of PCR-RFLP patterns and 16S rDNA sequences of unknown clostridial isolates with patterns and sequences of reference strains may not effect ready identification of these micro-organisms. The results of this study will be useful in diagnosis of the cause of premature spoilage of chilled vacuum-packed meats and in tracing spoilage-causing clostridia to their source(s) in the abattoir.
Asunto(s)
Clostridium/clasificación , Frío , Microbiología de Alimentos , Productos de la Carne/microbiología , Clostridium/genética , Clostridium/crecimiento & desarrollo , ADN Bacteriano/análisis , ADN Ribosómico/análisis , Embalaje de Alimentos , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , ARN Ribosómico 16S/análisis , Alineación de Secuencia , Análisis de Secuencia de ADN , VacioRESUMEN
Two strains of a psychrotolerant Clostridium, isolated from vacuum-packed, temperature-abused beef, were characterized using a multiphasic approach. The strains were Gram-positive motile rods producing elliptical subterminal spores during early stationary growth phase. The strains were psychrotolerant. At pH 7.0, they grew between 3.8 and 40.5 degrees C; their optimum growth temperature was 30.0-38.5 degrees C. At 30 degrees C, the pH range for growth was between 4.7 and 9.5; the optimum pH for growth was 6.4-7.2. The organisms were proteolytic and saccharolytic, lecithinase-positive and hydrolysed gelatin. The fermentation products formed in peptone/yeast extract/glucose/starch broth were acetate, ethanol, butyrate, isovalerate, butanol, isobutyrate, oxalacetate, lactate, hydrogen and carbon dioxide. The DNA G + C compositions of the two meat strains were 27.3 and 28.4 mol%. Phylogenetic analyses indicated that the strains belong to Cluster I of the genus Clostridium (sensu Collins et al., 1994). The new strains differed from phylogenetically related clostridia in terms of cellular fatty acid composition, soluble protein profiles and phenotypic properties. On the basis of phenotypic and genotypic characterization data, the strains were assigned to a new species for which the name Clostridium frigidicarnis is proposed; strain SPL77AT (= DSM 12271T) is the type strain.
Asunto(s)
Clostridium/clasificación , Microbiología de Alimentos , Embalaje de Alimentos , Carne/microbiología , Animales , Técnicas de Tipificación Bacteriana , Composición de Base , Bovinos , Clostridium/genética , Clostridium/aislamiento & purificación , Clostridium/fisiología , Frío , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Gases , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
The 16S rRNA gene from the Thermococcus New Zealand isolate AN1 was cloned and sequenced. Analysis of the gene revealed the presence of signature sequences, indicating that strain AN1 represents a new species of the genus Thermococcus. Since the isolate AN1 differed from other thermococci in both its lower optimal NaCl concentration and generally lower optimal temperature for growth, in its unusual lipid membrane composition, and in its sensitivity to antibiotics, we propose that strain AN1 represents a new species of Thermococcus. The proposed name is Thermococcus zilligii, and the type strain is DSM 2770.
Asunto(s)
Archaea/clasificación , Archaea/genética , ADN Ribosómico/genética , ARN Ribosómico 16S/genética , Técnicas de Tipificación Bacteriana , Clonación Molecular , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
We describe here the sequence and transcriptional analysis of a gene from the euryarchaeal Thermococcus species AN1 (DSM 2770) encoding a protein homologous to the alpha subunit of the eukaryal SRP receptor (SR alpha). The AN1 protein is found to share the highest degree of homology with the only other described archaeal SR alpha homolog characterized from the hyperthermophilic crenarchaeal Sulfolobus solfataricus. Sequence analysis of the translation of the AN1 gene reveals the presence of the following previously described domains: an N-terminal alpha domain rich in acidic and basic residues; an X domain and four GTP binding motifs (G1-G4). A putative guanine nucleotide dissociation stimulator binding element is also present. The AN1 SR alpha protein would now represent the shortest variant of this expanding family with 329 residues and a predicted molecular weight of 36.4 kDa. A Northern analysis indicates that the AN1 SR alpha protein gene transcript is present at low levels suggesting that SR alpha is likely to be only a minor cell constituent. The presence of an SR alpha homolog in another kingdom within the archaeal domain possessing the full suite of conserved motifs is significant in several respects. It not only supports the monophyletic character of the domain Archaea but suggests that these homologs have similar functions in these organisms and emphasises the ancient origins of the protein export machinery.
Asunto(s)
Archaea/genética , Genes Bacterianos , Receptores Citoplasmáticos y Nucleares/genética , Receptores de Péptidos/genética , Secuencia de Aminoácidos , Secuencia de Bases , Evolución Biológica , Transporte Biológico , Células Eucariotas , Código Genético , Datos de Secuencia Molecular , ARN Bacteriano/análisis , ARN Mensajero/análisis , Mapeo Restrictivo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Sulfolobus/genéticaRESUMEN
The han1A gene, encoding a subunit of the histone-like protein HAN1 from the Thermococcus species AN1, has been cloned and sequenced. Sequence analysis of the translation product of the gene demonstrates homology with other archaeal histone-like proteins of the 'HMf family' and eukaryal consensus sequences, particularly H4. The region of highest homology between the AN1 histone subunit, termed the HAN1A1 subunit, and the H4 consensus is suggested, by the 3-dimensional structure of the histone octamer, to interact with the minor groove of DNA. The results presented add further weight to the notion that the 'archaeal histones' and the eukaryal histones are indeed related and that the approximate 65 amino acid residue length of the archaeal histones represents the archaeal equivalent of the histone fold structural building block common to all eukaryal histones.
Asunto(s)
Archaea/química , Archaea/genética , Proteínas Arqueales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Genes Bacterianos , Histonas/química , Histonas/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Clonación Molecular , Secuencia de Consenso , Secuencia Conservada , Células Eucariotas , Datos de Secuencia Molecular , Biosíntesis de Proteínas , Conformación Proteica , Ribosomas/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , TATA Box , Transcripción GenéticaRESUMEN
We have purified and characterized the histone-like protein, termed HAN1, and an HAN1-associated DNA-binding protein (hDBP) from nucleoids of the hyperthermophilic Thermococcus-like AN1. HAN1 is shown to be composed of two subunits, to be thermally stable and to compact DNA in a reversible manner. The N-terminal sequence of HAN1 shares a high degree of homology with HMf, the histone-like protein from Methanothermus fervidus. Consistent with this, the toroidal wrapping of DNA by HAN1 resembles that described for HMf. However, significant differences in both twist and writhe components of these complexes are indicated by the 12.0 bp helical repeat produced during hydroxyl radical footprinting with HAN1. Furthermore, the increased stability of HAN1: DNA complexes allows DNA to be protected from thermal denaturation and cleavage by the restriction enzyme TaqI at 65 degrees C. The hDBP, which co-purified with HAN1,is shown to represent a major portion of the acid-washed nucleoid protein in AN1 and to enhance the mobility of DNA directly, yet decrease the mobility of HAN1:DNA complexes.
Asunto(s)
Archaea/química , Proteínas Arqueales , Proteínas Bacterianas/aislamiento & purificación , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/aislamiento & purificación , Histonas/aislamiento & purificación , Secuencia de Aminoácidos , Archaea/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Huella de ADN , ADN Bacteriano/química , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Histonas/química , Histonas/metabolismo , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Desnaturalización de Ácido Nucleico , TemperaturaRESUMEN
HMf, a histone from the hyperthermophilic archaeon Methanothermus fervidus binds double-stranded DNA molecules in vitro, forming compact structures that visibly resemble eukaryal nucleosomes. We show here that HMf binding increases the helical periodicity of DNA molecules to approximately 11 base pairs (bp) per turn and that DNA molecules in these nucleosome-like structures are constrained in positive toroidal supercoils. Based on the mass of HMf needed to cause a change in linking number (delta Lk), the maximum delta Lk introduced into circular DNA molecules of known sizes, and electron microscopy, we estimate that each HMf-DNA structure contains between 90 and 150 bp of DNA wrapped in 1.5 positive toroidal supercoils around a core of four HMf molecules. A model and pathway for the formation of these structures in vitro are presented and the possible role of positive toroidal wrapping of the M. fervidus genome in vivo is discussed.
Asunto(s)
Proteínas Arqueales , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/metabolismo , Euryarchaeota/metabolismo , Histonas/metabolismo , Plásmidos , Secuencia de Bases , Sitios de Unión , ADN Circular/genética , ADN Circular/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Radicales Libres , Hidróxidos , Radical Hidroxilo , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Conformación ProteicaRESUMEN
Exogenous thymine was found to be taken up very slowly by Pseudomonas aeruginosa in comparison to other pyrimidines, and most of it was catabolized by the cell. The existence of a functional, although inefficient, thymine salvage pathway was demonstrated and this pathway operated more effectively when de novo thymidine nucleotide biosynthesis was inhibited by trimethoprim or methotrexate. The mechanism of thymine salvage by P. aeruginosa appears to be different from that of Escherichia coli and Pseudomonas acidovorans as thymidine was not incorporated into the DNA. Like P. acidovorans, P. aeruginosa lacked thymidine phosphorylase activity. Unsuccessful attempts were made to isolate thymine auxotrophs.