New species of bacteria in the genus Kurthia--Kurthia sibirica sp. nov

Six aerobic gram-positive nonspore-forming bacterial strains belonging to the Kurthia genus were isolated from the Magadan (Susuman) mammoth found in the permafrost of the East Siberia. The strains are a phenotypically homogeneous group different from the two known species (K. zopfii and K. gibsonii) in requiring more vitamins, the absence of growth in a medium with 7% NaCl, and a low level of DNA-DNA hybridization (not more than 45%). Moreover, the strains differ from K. zopfii in the synthesis of a yellow pigment, the activity of phosphatase, and the absence of coccoid forms; the bacteria differ from K. gibsonii in the absence of growth at a temperature above 40 degrees C. The organisms are referred to as Kurthia sibirica sp. nov. The type strain 13-2 has been deposited in the All-Union Collection of Microorganisms as strain VKB B-1549.     

Fatty Acid and Polar Lipid Composition in the Classification of Kurthia

Strains of Kurthia zopfii were degraded by acid methanolysis and the non-hydroxylated fatty acid esters so released were examined by gas liquid chromatography. The major fatty acid types were straight-chain, anteiso - and iso -methyl branched-chain acids. Monounsaturated fatty acids were not detected. The major fatty acid in five of the six strains examined consisted of 12-methyltetra-decanoic ( anteiso -C₁₅) acid. The other strain possessed major amounts of both 13-methyltetradecanoic ( iso -C₁₅) and anteiso -C₁₅ acids. Polar lipids of all the strains were examined by two-dimensional thin-layer chromatography. All possessed a very simple polar lipid composition consisting of diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine.     

Action of Bacterial Growth on the Sarcoplasmic and Urea-Soluble Proteins from Muscle: I. Effects of Clostridium perfringens, Salmonella enteritidis, Achromobacter liquefaciens, Streptococcus faecalis, and Kurthia zopfii

Comparisons of the starch-gel patterns of uninoculated aseptic control samples from rabbit and pig muscle with similar samples inoculated and incubated with Clostridium perfringens, Salmonella enteritidis, Achromobacter liquefaciens, and Kurthia zopfii were made. Results indicated that C. perfringens caused extensive alteration in the proteins or enzymes, or both, of the sarcoplasmic fraction of porcine muscle, whereas S. enteritidis and S. faecalis caused complete breakdown of only myoglobin. Neither A. liquefaciens nor K. zopfii showed any measurable amount of proteolysis in the sarcoplasmic fraction from pig muscle. Although some of the bands in the starch-gel pattern of rabbit muscle decreased in size and intensity of staining, complete proteolysis of any protein fraction was absent for all test organisms. The disc-gel patterns of the 8 m urea-soluble proteins showed that C. perfringens caused extensive proteolysis in pig muscle and a lesser extent of proteolysis in rabbit muscle. None of the other organisms utilized in this study had any measurable effect upon the urea-soluble proteins. In addition, a simple procedure for aseptic isolation of muscle samples for studying meat spoilage is outlined. Results indicate that careful sanitation and cleanliness will give suitable samples for meat spoilage investigations.     

Numerical taxonomy of certain coryneform bacteria

da Silva, G. A. Nigel (Iowa State University, Ames), and John G. Holt. Numerical taxonomy of certain coryneform bacteria. J. Bacteriol. 90:921-927. 1965-An electronic computer was used to sort 32 strains of coryneforms into groups with the tree-sort program. The similarity values obtained in this procedure were then used to construct a dendrogram depicting the phenetic resemblance among the taxa. The results indicated that all the phytopathogens studied were sufficiently distinct from the type species, Corynebacterium diphtheriae, to be excluded from the genus Corynebacterium. The grouping of some of the phytopathogens with Microbacterium lacticum is discussed. C. fascians appeared so distinct from the other strains studied that it should probably be excluded from the Corynebacteriaceae. The phenetic resemblance of Brevibacterium linens and Arthrobacter globiformis was emphasized, and the new combination, Arthrobacter linens, was proposed. In addition, because of distinct dissimilarity from the type species, it would seem desirable to exclude Arthrobacter tumescens from the genus Arthrobacter. The justification for classifying Kurthia zopfii in a family separate from the Corynebacteriaceae would appear to be open to serious question. It was concluded that the present taxonomic positions of Listeria monocytogenes, Cellulomonas biazotea, and Cellulomonas fimi are satisfactory.     

Numerical classification of some Rhodococci, Corynebacteria and related organisms

Nineteen strains of Corynebacterium sensu stricto, 23 received as Corynebacterium equi or Rhodococcus equi, marker cultures of Arthrobacter, Brevibacterium, Bacterionema matruchotii, Cellulomonas flavigena, Kurthia zopfii, Listeria denitrificans, Microbacterium lacticum, Rhodococcus rubropertinctus and 88 representatives of Mycobacterium, Nocardia, Rhodococcus and the 'aurantiaca' taxon were the subject of numerical phenetic analyses using 92 characters. The data were examined using the simple matching (SSM) and Jaccard (SJ) coefficients and clustering was achieved using the average linkage algorithm. With a single exception, strains containing meso-diaminopimelic acid, arabinose, galactose and mycolic acids were recovered in five aggregate clusters corresponding to Corynebacterium sensu stricto, Mycobacterium, Nocardia, Rhodococcus and the 'aurantiaca' taxon. Most of the Corynebacterium (Rhodococcus) equi strains formed a good taxospecies which included the type strain of Corynebacterium hoagii. The numerical data, and the results of earlier chemical and genetical studies, also provide sufficient evidence for the transfer of Bacterionema matruchotii to Corynebacterium sensu stricto as Corynebacterium matruchotii comb.nov. and for the recognition of Rhodococcus globerulus sp.nov. for some strains previously classified as Rhodococcus rubropertinctus (Hefferan) Goodfellow & Alderson. The classification of the remaining marker strains correlates well with other major developments in coryneform taxonomy.     

Suppression of leaf feeding and oviposition of phytophagous ladybird beetles (Coleoptera: Coccinellidae) by chitinase gene-transformed phylloplane bacteria and their specific bacteriophages entrapped in alginate gel beads

The chitinase gene-transformed strain KPM-007E/chi of Enterobacter cloacae was vitally entrapped in sodium alginate gel beads with its specific virulent bacteriophage EcP-01 to provide a new method for microbially digesting chitinous peritrophic membranes of phytophagous ladybird beetles Epilachna vigintioctopunctata. First, chitinase SH1 from a gram-positive bacterium Kurthia zopfii was overproduced by Escherichia coli cells and purified by affinity column chromatography. The purified enzyme effectively digested peritrophic membranes dissected from the ladybird beetles to expose epithelial tissues beneath the peritrophic membrane, and the beetles that had ingested chitinase after submergence in chitinase solution had considerably reduced their feeding on tomato leaves. KPM-007E/chi, entrapped in the alginate beads, released the chitinase. More chitinase was released when KPM-007E/chi was present with their specific virulent bacteriophage EcP-01 in the beads because of lysis of bacterial cells infected with the bacteriophages. This chitinase release from the microbial beads (containing KPM-007E/chi and EcP-01) was sufficient to digest the peritrophic membrane as well as to suppress feeding of bead-sprayed tomato leaves by the ladybird beetles. A daily supply of tomato leaves treated with the microbial beads considerably suppressed leaf feeding and oviposition by the ladybird beetles, suggesting a possible application of chitinase-secreting bacteria for suppressing herbivorous insect pests.     

Effect of Packaging under Carbon Dioxide, Nitrogen or Air on the Microbial Flora of Pork Stored at 4°C

Changes in the microbial flora of pork packed in laminated plastic bags and stored at 4 °C were studied in an initial atmosphere of carbon dioxide, nitrogen or air. The time needed for the total aerobic count at 28 °C to reach 5 × 10⁶ organisms/cm² was about 7 times longer in carbon dioxide than in air, whilst in nitrogen it was about twice as long.
The predominant organisms on fresh pork taken directly from the processing line were: Acinetobacter calcoaceticus , non-fluorescent Pseudomonas spp. and Flavobacterium spp. After storage in air for 7 d, more than 90% of the flora consisted of non-fluorescent Pseudomonas spp. After storage in nitrogen for 10 d, 70% of the flora consisted of non-fluorescent Pseudomonas spp. with lower levels of fluorescent Pseudomonas spp., Kurthia zopfii, Aeromonas hydrophila and Lactobacillus plantarum. The non-fluorescent Pseudomonas spp. could be divided into three different groups, on proteolytic and lipolytic ability; the distribution of the groups was markedly different between pork loins stored in air and nitrogen.
On pork stored in carbon dioxide for 21 d the flora consisted of L. plantarum together with lower levels of heterofermentative lactic acid bacteria. When the storage time in carbon doxide was prolonged to 35 d, the proportion of heterofermentative lactic acid bacteria increased to about 50% of the flora.     

Growth of Prototheca isolates on n-hexadecane and mixed-hydrocarbon substrate

Prototheca zopfii, an achlorphyllous alga, was capable of using hydrocarbons as sole carbon and energy source. The ability of P. zopfii to use hydrocarbons did not correlate with source of isolation. Seventy-five percent of the P. zopfii cultures recovered from sewage, plants, or animals utilized hydrocarbons. Other Prototheca species and P. zopfii that did not utilize hydrocarbons were isolated simultaneously from several sources with isolates that did use hydrocarbons. Species type rather than source of isolation was the predominant factor that determined hydrocarbon utilization.     

Growth of Prototheca isolates on n-hexadecane and mixed-hydrocarbon substrate.

Prototheca zopfii, an achlorphyllous alga, was capable of using hydrocarbons as sole carbon and energy source. The ability of P. zopfii to use hydrocarbons did not correlate with source of isolation. Seventy-five percent of the P. zopfii cultures recovered from sewage, plants, or animals utilized hydrocarbons. Other Prototheca species and P. zopfii that did not utilize hydrocarbons were isolated simultaneously from several sources with isolates that did use hydrocarbons. Species type rather than source of isolation was the predominant factor that determined hydrocarbon utilization.     

A ribosomal DNA fragment of Listeria monocytogenes and its use as a genus-specific probe in an aqueous-phase hybridization assay.

cDNAs were prepared from the total RNA of Listeria monocytogenes ATCC 19118 and used as probes to screen a genomic library of the same strain. Four clones were identified which contained ribosomal DNA fragments. Recombinant DNA from one of them was fractionated and differentially hybridized with the cDNA probes to RNA of L. monocytogenes and Kurthia zopfii. The resulting hybridization pattern revealed an HpaII fragment of 0.8 kb that was specific for the L. monocytogenes strain. The nucleotide sequence of this fragment showed 159 bases of the 3' end of the 16S rRNA gene, 243 bases of the spacer region, and 382 bases of the 5' end of the 23S rRNA gene. In dot blot hybridization assays, the 32P-labeled 784-bp fragment was specific only for Listeria species. Dot blot assays revealed that the 32P-labeled fragment can easily detect > or = 10 pg of total nucleic acids from pure cultures of L. monocytogenes, which corresponds to approximately 300 bacteria. This fragment was also used as a probe in an assay named the heteroduplex nucleic acid (HNA) enzyme-linked immunosorbent assay. In this system, the biotinylated DNA probe is hybridized in the aqueous phase with target RNA molecules and then specific HNAs are captured by HNA-specific antibodies. Captured HNA molecules are revealed with an enzyme conjugate of streptavidin. In a preliminary HNA enzyme-linked immunosorbent assay, the 784-bp fragment maintained its specificity for Listeria spp. and could detect 5 x 10(2) cells in artificially contaminated meat homogenate.     

Susceptibility and features of the ultrastructure of Prototheca zopfii following exposure to copper sulphate,silver nitrate and chlorexidine

One of the most important forms of the occurrence of protothecosis is bovine mastitis. Studies on the "in vivo" and "in vitro" susceptibility to antimicrobials have shown that the microorganism is resistant to most of them. Looking for alternative treatments this study aimed to study the susceptibility to copper sulphate (which has an important algicide effect) and silver nitrate (used in dairy cattle breeding for the cauterization of mammary glands) and also to chlorexidine (an important post-dipping anti-septic used in dairy practice), and the effect of these antimicrobials in the ultrastructure of Prototheca zopfii before and after the exposure to these drugs. The "in vitro" susceptibility tests to chlorexidine, silver nitrate and copper sulphate of the strains of Prototheca zopfii for the determination of their minimal microbicidal concentrations (MMC), were performed using the tube dilution method in Sabouraud dextrose broth and evaluation of colony growth after plating in Sabouraud dextrose agar. The MMCs of chlorexidine, copper sulphate and silver nitrate of the 50 strains tested were 0.01%, 0.1% and 0.3%, respectively. The tubes containing the material used in the antimicrobial susceptibility tests were prepared for the examination in an electron microscope. The untreated controls of P. zopfii showed a similar ultrastructural appearance with the typical characteristics of the microorganism. Cells exposed to silver nitrate showed changes suggesting thickness of the cell wall. Cells exposed to chlorexidine showed changes suggesting degradation of intra-cellular organelles present in the cytoplasm. P. zopfii treated with copper sulphate showed changes suggesting fibrilation of inner layer of cell wall.     

Cloning and characterization of biotin biosynthetic genes of Kurthia sp

The biotin biosynthesis genes of Kurthia sp., which is an aerobic gram-positive bacterium, were cloned from Kurthia sp. 538-KA26 and characterized. Eleven biotin biosynthetic genes have been identified in Kurthia sp. Kurthia sp. has two genes coding for KAPA synthase, bioF and bioFII, and also has two genes coding for BioH protein, bioH and bioHII. In addition, three genes, orf1, orf2, and orf3, whose functions are unknown, were found in the biotin gene clusters of Kurthia sp. The bioA, bioD, and orf1 genes are arranged in a gene cluster in the order orf1bioDA, and the bioB, bioF, and orf2 genes are arranged in a gene cluster in the order orf2bioFB. These gene clusters proceed to both directions; the face to face promoters and two 40-bp of palindrome sequences exist upstream of the orf1 and orf2 genes. The bioC, bioFII, and bioHII genes are arranged in a gene cluster in the order bioFIIHIIC; a 40-bp of palindrome sequence exists upstream of the bioFII gene. The bioH and orf3 genes are arranged in a gene cluster in the order bioHorf3; a palindrome sequence was not found upstream of the bioH gene. These palindrome sequences are extremely similar to each other, suggesting that the orf1bioDA, orf2bioFB, and bioFIIHIIC gene clusters are regulated by biotin. Kurthia sp. does not have the bioW gene coding pimeloyl-CoA synthase, suggesting that pimeloyl-CoA may be produced by a different pathway than that of gram-positive bacterium B. subtilis or B. sphaericus, further suggesting a modified fatty acid synthesis pathway via acetyl-CoA instead as E. coli has.     

Effect of Growth Rate on the Macromolecular Composition of Prototheca zopfii, a Colorless Alga Which Divides by Multiple Fission

Prototheca zopfii, a eukaryote that divides by multiple fission, was investigated to determine how growth rate controls daughter cell number. The macromolecular composition, cell size, and number of nuclei per cell were determined in cultures during balanced growth in various media. Cellular mass, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), carbohydrate, and nuclear number increased as positive linear functions of growth rate, whereas nuclear ploidy remained constant with a value of 0.098 pg of DNA/nucleus. The ratios of RNA to protein, protein to mass, and carbohydrate to mass were unaffected by growth rate, whereas the ratios of DNA to protein and RNA to DNA could be expressed as curvilinear functions of growth rate, the former negative and the latter positive. The dependency of normalized gene dosage (DNA/protein) on growth rate appeared as a distinguishing feature of multiple fission. Determination of the normalized rates of protein and RNA synthesis revealed that both increase linearly with growth rate. It is concluded that Prototheca zopfii may exist in a number of physiological states which are characterized by a unique size and macromolecular composition and which are dictated by growth rate.     

DEGRADATION OF ADENINE BY PROTOTHECA ZOPFII (CHLOROPHYTA)1

The purines 6-amino-2-hydroxypurine and 6-amino-8-hydroxypurine, not normally associated with purine degradation in algae, were isolated by high-pressure liquid chromatography from cell extracts of Prototheca zopfii Krüger grown with adenine as the sole nitrogen source .     

Degradation of Petroleum by an Alga, Prototheca Zopfii

Prototheca zopfii is an achlorophyllous alga which degrades oil. It has been found to degrade 10 and 40% of a motor oil and crude oil, respectively, when tested under appropriate conditions. Degradation of the crude oil observed in this study compares well with the amount of degradation accomplished by bacteria. P. zopfii was found to degrade a greater percentage of the aromatic hydrocarbons in motor oil than of the saturated hydrocarbons and a greater percentage of saturated hydrocarbons in crude oil than of aromatic hydrocarbons. Resins and asphaltens were produced during degradation of motor oil, whereas these fractions in crude oil were degraded. P. zopfii did not demonstrate preferential utilization of lower homologues of cycloalkanes and aromatics as has been observed with bacteria.     

<Emphasis Type="Italic">Kurthia ruminicola</Emphasis> sp. nov., isolated from the rumen contents of a Holstein cow

Gram-staining-positive, motile, rod-shaped bacteria, designated as H31022^T and H31024 was isolated from rumen contents of a Holstein cow. Optimum growth occurred at 25°C and pH 7.0 on R2A agar medium. Oxidase and catalase activities are positive. The 16S rRNA gene sequence (1,452 bp) of the new isolates revealed they belong to the genus Kurthia of the phylum Firmicutes. Highest gene sequence similarities were assessed to be with Kurthia massiliensis JC30^T (98.4%), Kurthia senegalensis JC8E^T (97.5%), and Kurthia populi 10y-14^T (97.4%). Kurthia sibirica DSM 4747^T (97.3%), Kurthia zopfii NBRC 101529^T (97.0%), and Kurthia gibsonii NCIMB 9758^T (96.7%). DNA G + C content of strains H31022^T and H31024 were 34.4% and 39.7%. Strains H31022^T and H31024 has the following chemotaxonomic characteristics; the major fatty acids are iso-C_15:0, iso-C_14:0 and anteiso-C₁₅; polar lipid profile contained diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), unknown aminophospholipids (APL), unknown glycolipids (GL), unknown phospholipids (PL), and unknown polar lipids (L); the major quinone is MK-7. Based on polyphasic taxonomic analysis, strains H31022^T (= KCTC 33923^T = JCM 19640^T) and H31024 (= KCTC 33924^T = JCM 19641^T) identified a novel species in the genus Kurthia for which the name Kurthia ruminicola sp. nov. is proposed.     

Isolation of Prothoteca zopfii from a case of bovine mastitis in Brazil

The isolation of Prothoteca zopfii, an algae lacking chlorophyll, from bovine mastitic milk, is described herein. The isolation was performed on 8% sheep blood agar, following incubation at 37 °C for 48 h. Based on biochemical tests, susceptibility to clotrimazole, and light and electron microscopic observation of cellular morphology the algae was identified as P. zopfii . The affected animal did not improve following treatment and had to be eliminated. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cutaneous Protothecosis in Patient with Diabetes Mellitus and Review of Published Case Reports

Protothecosis is an opportunistic infection caused by Prototheca, usually called as saprophytes, and is frequently found in natural and living surroundings with low virulence, but may cause chronic infection in immunocompromised individuals. We report a case of cutaneous protothecosis with zopfii var. portoricensis infection in a 66-year-old diabetic woman following hand surgery on middle right finger. Mycology study showed that smooth, creamy white, yeast-like colonies grown after necrotic tissue was cultured on Sabouraud dextrose agar at both 37 and 25°C. The organism was then identified as Prototheca zopfii var. portoricensis by molecular identification and also found from histopathology of the lesion. The lesion got improved with intravenous amphotericin B and itraconazole.     

无绿藻的表型及其分子生物学鉴定

目的通过表型及分子生物学方法,正确鉴定无绿藻及其变种。方法回顾、总结与分析无绿藻的形态与结构、表型特征及生理生化及分子生物学鉴定、对常见抗真菌药物的敏感性及组织病理学特征。结果通过表型及分子生物学技术成功鉴定2株来自脑脊液及淋巴结的无绿藻:中型无绿藻碳水化合物变种及中型无绿藻波多黎各变种,并展示相关照片。结论无绿藻病的症状尚无特异性,其诊断主要依靠真菌学检查。标本的直接镜检、真菌培养及组织病理检查是主要手段。对无绿藻菌种的鉴定除了菌落形态、镜下结构外(含内孢子的孢子囊是无绿藻属的重要特征),糖类、醇类的同化利用,温度试验,结合分子生物学鉴定将有助于菌种的鉴定。     

A new species of aspergillus fumigatus group and comments upon its synonymy

Prototheca zopfii was isolated repeatedly from the nails of a patient. The patient had preexisting nail disease and colonization by the alga was opportunistic.