Inactivation of Nocardiophages φC and φEC by Extracts of Bacteriophage-Attachable Cells

Cultures of several species of Nocardia, including N. erythropolis Mat-Ce and Mat-cE mating strains, were extracted with solvents in an attempt to isolate an inactivating complex for nocardiophages phiC and phiEC. Ethanol was the only solvent found effective in solubilizing an inhibitory substance. Inactivating extracts were obtained from the cells of all species to which the phage were able to attach. After extraction of whole cells or cell wall preparations, the phage could not effectively attach to them. Both phages phiC and phiEC were inactivated by the same complex. However, phage phiEC inactivation was 10-fold greater than phiC inactivation. The velocity of inactivation was about 4.1 x 10(2) plaque-forming units per microgram per minute for phiC and 1.1 x 10(3) plaque-forming units per microgram per minute for phage phiEC. The cell extracts required divalent cations for phage inactivation. The inhibitory capacity of the cell extracts was reduced or lost by the activity of proteolytic enzymes, Tween 80, 2-mercaptoethanol, thymol, and sodium lauryl sulfate. Boiling the extract for 10 min did not alter its activity. The inactivating substance was postulated to be a lipoprotein of considerable complexity, unique in the ease with which it is solubilized from host cells by ethanol.     

Excision of bacteriophage lambda from a site in the arabinose B gene.

A lambda lysogen with the prophage inserted into the arabinose B gene of Escherichia coli strain K-12 has been prepared. Induction of the phage from this lysogen yields viable phage at a frequency 4 X 10(-6) that found for induction of lysogens with phage inserted at the normal attachment site. Over 30% of the phage particles induced from the insertion in ara are arabinose-transducing phage. The excision end points of 62 independently isolated, nondefective araC-transducing phage containing less than the entire araC gene were genetically determined and were found to be randomly distributed through the araC gene. The amount of arabinose deoxyribonucleic acid contained on four selected transducing phage was determined by electron microscopy of deoxyribonucleic acid heteroduplexes, providing a physical map of the araC gene. The efficiency with which these phage transduce araC and araB point mutations was found to be approximately proportional to the homology length available for recombination.     

Minicell production and bacteriophage superinducibility of thymidine-requiring strains of Haemophilus influenzae.

Aminopterin- or trimethoprin-resistant thymidine-requiring strains of Haemophilus influenzae produce minicells, and the ratio of minicells to cells increases during the stationary phase of growth. Strain LB11, isolated after mutagenesis of a thymidine-requiring strain (Rd thd), produces more minicells than the parent strain. The mutations involved in high frequency minicell production have been transferred into the wild type (strain Rd) by transformation. The thymidine requirement in the resulting strain, MCl, is essential for minicell production, since spontaneous revertants of MCl to prototrophy do not produce minicells. The ratio of minicells to cells was increased more than 10(3)-fold by differential centrifugation. The minicells contain little or no deoxyribonucleic acid (DNA). Phage HPlcl apparently cannot attach to minicells. Competent cells of LB11 and its thymidine-requiring parent strain produce defective phage as a result of exposure to transforming DNA, whereas only LB11 produces many defective phage in response to the competence regime alone. Competent HP1c1 and S2 lysogens of MC1 and Rd thd are also superinducible by transforming DNA, but competent LB11 lysogens produced about the same amount of HP1c1 or S2 phage with or without exposure to transforming DNA possibly because of competition between the induced defective phage and Hp1c1 or S2 phage.     

Biodesulfurization of benzothiophene and dibenzothiophene by a newly isolated Rhodococcus strain

Rhodococcus sp. KT462, which can grow on either benzothiophene (BT) or dibenzothiophene (DBT) as the sole source of sulfur, was newly isolated and characterized. GC and GC-MS analyses revealed that strain KT462 has the same BT desulfurization pathway as that reported for Paenibacillus sp. A11-2 and Sinorhizobium sp. KT55. The desulfurized product of DBT produced by this strain, as well as other DBT-desulfurizing bacteria such as R. erythropolis KA2-5-1 and R. erythropolis IGTS8, was 2-hydroxybiphenyl. A resting cells study indicated that this strain was also able to degrade various alkyl derivatives of BT and DBT.     

Characterization of the genome of the polyvalent lytic bacteriophage GTE2, which has potential for biocontrol of Gordonia-, Rhodococcus-, and Nocardia-stabilized foams in activated sludge plants

Hydrophobic Actinobacteria are commonly associated with the stabilization of foams in activated sludge systems. One possible attractive approach to control these foam-stabilizing organisms is the use of specific bacteriophages. We describe the genome characterization of a novel polyvalent DNA phage, GTE2, isolated from activated sludge. This phage is lytic for Gordonia terrae, Rhodococcus globerulus, Rhodococcus erythropolis, Rhodococcus erythropolis, Nocardia otitidiscaviarum, and Nocardia brasiliensis. Phage GTE2 belongs to the family Siphoviridae, possessing a characteristic icosahedral head encapsulating a double-stranded DNA linear genome (45,530 bp) having 10-bp 3'-protruding cohesive ends. The genome sequence is 98% unique at the DNA level and contains 57 putative genes. The genome can be divided into two components, where the first is modular and encodes phage structural proteins and lysis genes. The second is not modular, and the genes harbored there are involved in DNA replication, repair, and metabolism. Some have no known function. GTE2 shows promising results in controlling stable foam production by its host bacteria under laboratory conditions, suggesting that it may prove useful in the field as a biocontrol agent.     

Relationship Between Prophage Induction and Transformation in Haemophilus influenzae

The interaction between transformation and prophages of HP1c1, S2, and a defective phage of Haemophilus influenzae has been investigated by measurement of (i) the effect of prophage on transformation frequency and (ii) the effect of transformation on phage induction. The presence of any of the prophages does not appreciably alter transformation frequencies in various Rec(+) and Rec(-) strains. However, exposure of competent lysogens to transforming deoxyribonucleic acid (DNA) may induce phage but only in Rec(+) strains, which are able to integrate transforming DNA into their genome. Transformation of Rec(+) lysogens with DNA irradiated with ultraviolet (UV) light causes the production of even more phage than results from unirradiated DNA, but this indirect UV induction is not as effective as direct induction by UV irradiation of lysogens. Both types of UV induction are influenced by the repair capacity of the host. Wild-type cells contain a prophage and can be induced by transformation to produce a defective phage, which kills a small fraction of the cells. Defective phage in wild-type cells are also induced by H. parainfluenzae DNA, and a much larger fraction of the cells is killed. Strain BC200, which is highly transformable but is not inducible for defective phage, is not killed by H. parainfluenzae DNA, suggesting that wild-type cells are killed by killed by this DNA because of phage induction. A minicell-producing mutant, LB11, has been isolated. Some phage induction occurs in this strain when the cells are made competent, unlike the wild type. A large majority of LB11 cells surviving the competence regime are killed by exposure to transforming DNA.     

Genetic analysis of spo0A and spo0C mutants of Bacillus subtilis with a phi 105 prophage merodiploid system.

An 8.0-kilobase chromosomal fragment of Bacillus subtilis which contained an intact spo0A gene was recloned onto temperate phage phi 105 from the rho 11dspo0A+-1 transducing phage. A specialized transducing phage, phi 105-dspo0A+-1, was constructed and used to transduce the spo0A12 mutant strain 1S9. A Spo+ transductant which was a single lysogen of the phi 105dspo0A+-1 transducing phage was isolated. From competent cells of this Spo+ transductant was isolated a Spo- (Spo0A) strain which was immune to phi 105. It was used to prepare a lysate of the phi 105dspo0A12 phage. Transduction of the spo0C9V recE4 strain with the phi 105dspo0A12 and phi 105dspo0A+-1 phages was carried out. The phi 105dspo0A+-1 phage gave rise to a large number of heat-resistant cells, but the phi 105dspo0A12 phage formed no heat-resistant cells. These results indicate that the spo0A12 and spo0C9V mutant genes do not complement each other in the ability to sporulate and that the spo0C9V mutation is located within the spo0A gene. Although the spo0C9V strain was completely asporogenous, the spo0C9V/spo0C9V diploid strain produced heat-resistant cells at a frequency of ca. 10(-3) in the sporulation medium. This result indicates that two copies of the spo0C9V mutant gene partially restore the ability of these cells to sporulate.     

High-frequency interconversion of turbid and clear plaque strains of bacteriophage f1 and associated host cell death

Under normal cultivation conditions, a mixture of turbid and clear plaques is often apparent in cultures of bacterial cells infected with filamentous bacteriophages. Beginning with a culture of wild-type filamentous phage f1, which itself produces turbid plaques, a clear plaque strain (c1) was isolated. From c1, the turbid plaque strain t1 was isolated; from t1, the clear plaque strain c2 was isolated; and from c2, the turbid plaque strain t2 was isolated. Each of these strains was generated with a frequency of approximately 1 x 10(-4). Although filamentous phages have been thought not to induce host cell death, both turbid and clear plaque strains of f1 killed host bacteria. Plating of bacterial cells 1 h after infection revealed that colonies produced by cells infected with either wild-type f1 or strain c2 were smaller than those derived from uninfected cells, and that colony formation by infected cells was reduced by 15% and 38%, respectively. The time course of bacterial growth revealed that, at 4 h after infection, the number of CFU per milliliter of culture of cells infected with wild-type f1 or with strain c2 was reduced by 27% and 95%, respectively, compared with that for uninfected cells. Microculture analysis also revealed that the percentages of nondividing cells in f1 or c2 infected were 19% and 52%, respectively, 4 h after infection with wild-type f1 or with strain c2; no such cells were detected in cultures of uninfected cells. Negative staining and electron microscopy showed that 20% and 61% of cells infected with wild-type f1 or with strain c2 were dead 4 h postinfection. Finally, although the rates of DNA synthesis were similar for infected and uninfected cells, the rates of RNA and protein synthesis were markedly reduced in infected cells.     

The highly virulent 2006 Norwegian EHEC O103:H25 outbreak strain is related to the 2011 German O104:H4 outbreak strain

In 2006, a severe foodborne EHEC outbreak occured in Norway. Seventeen cases were recorded and the HUS frequency was 60%. The causative strain, Esherichia coli O103:H25, is considered to be particularly virulent. Sequencing of the outbreak strain revealed resemblance to the 2011 German outbreak strain E. coli O104:H4, both in genome and Shiga toxin 2-encoding (Stx2) phage sequence. The nucleotide identity between the Stx2 phages from the Norwegian and German outbreak strains was 90%. During the 2006 outbreak, stx(2)-positive O103:H25 E. coli was isolated from two patients. All the other outbreak associated isolates, including all food isolates, were stx-negative, and carried a different phage replacing the Stx2 phage. This phage was of similar size to the Stx2 phage, but had a distinctive early phage region and no stx gene. The sequence of the early region of this phage was not retrieved from the bacterial host genome, and the origin of the phage is unknown. The contaminated food most likely contained a mixture of E. coli O103:H25 cells with either one of the phages.     

Heat Induction of Prophage φ105 in Bacillus subtilis: Replication of the Bacterial and Bacteriophage Genomes

A temperature-inducible mutant of temperate Bacillus bacteriophage phi105 was isolated and used to lysogenize a thymine-requiring strain of Bacillus subtilis 168. Synthesis of phage and bacterial deoxyribonucleic acid (DNA) was studied by sucrose gradient centrifugation and density equilibrium centrifugation of DNA extracted from induced bacteria. The distribution of DNA in the gradients was measured by differential isotope and density labeling of DNA before and after induction and by measuring the biological activity of the DNA in genetic transformation, in rescue of phage markers, and in infectivity assays. At early times after induction, but after at least one round of replication, phage DNA remains associated with high-molecular-weight DNA, whereas, later in the infection, phage DNA is associated with material of decreasing molecular weight. Genetic linkage between phage and bacterial markers can be demonstrated in replicated DNA from induced cells. Prophage induction is shown to affect replication of the bacterial chromosome. The overall rate of replication of prelabeled bacterial DNA is identical in temperature-induced lysogenics and in "mock-induced" wild-type phi105 lysogenics. The rate of replication of the bacterial marker phe-1 (and also of nia-38), located close to the prophage in direction of the terminus of the bacterial chromosome, is increased in induced cells, however, relative to other bacterial markers tested. In temperature-inducible lysogenics, where the prophage also carries a ts mutation which blocks phage DNA synthesis, replication of both phage and bacterial DNA stops after about 50% of the phage DNA has replicated once. The results of these experiments suggest that the prophage is not initially excised in induced cells, but rather it is specifically replicated in situ together with adjacent parts of the bacterial chromosome.     

Use of claydite-immobilized oil-oxidizing microbial cells for purification of water from oil

Oil-oxidizing bacteria were isolated from oil-polluted soil and water samples and identified as Acinetobacter calcoaceticus K-4, Nocardia vaceinii K-8, Rhodococcus erythropolis EK-1, and Mycobacterium sp. K-2. It was found that immobilization of the bacteria on an expanded clay aggregate accelerated their growth and consumption of hydrocarbon substrates. It was also found that water polluted with 100 mg/l oil could be purified with Rhodococcus erythropolis EK-1 and Nocardia vaceinii K-8 cells immobilized in this way. The dependence of the degree of water purification on its flow rate, aeration, and availability of nitrogen and phosphorus sources was determined. The efficiency of water purification from oil by immobilized Rhodococcus erythropolis EK-1 cells at high flow rates (of up to 0.68 l/h), low aeration (of 0.1 l/l per min) and an intermittent supply of 0.01% diammonium phosphate reached 99.5-99.8%.     

Properties of a Salmonella typhimurium Mutant with an Incomplete Deficiency of Uridinediphosphogalactose-4-Epimerase

A galactose-negative mutant, nonleaky in respect to fermentation and utilization, isolated from a smooth Salmonella typhimurium strain by phage selection and inferred deficient of uridine diphosphate (UDP)-galactose-epimerase, was used for experiments on relation of somatic lipopolysaccharide (LPS) character to virulence. Extracts of induced mutant cells retained ca. 1% of wild-type epimerase activity and had only ca. 5% of wild-type kinase and uridyl transferase activities; also, some cultural properties of the mutant differed from those of mutants with complete defects of epimerase only. The mutant was not galactose sensitive, presumably because of its kinase defect. Although the mutant had the phage pattern (including C21-sensitivity) of an epimerase mutant, it was susceptible to transduction by phage P22 and was O-agglutinable, even when grown on defined medium; its LPS must therefore contain some O polymer, including endogenous galactose, resulting from residual epimerase activity. Growth on galactose-supplemented medium restored smooth phage sensitivity; since the mutant was partly inducible this may result, at least in part, from increased endogenous production of UDP-galactose. The mutant was made galactose positive by introduction of an F'-gal(+) plasmid. Base-change and frame-shift mutagens did not increase the frequency of reversion above the spontaneous rate. An insertion into the operator-promoter region of the gal operon seems the most likely mechanism of the mutation.     

Thymineless bacteriophage induction in Staphylococcus aureus. I. High-frequency transduction with lysates containing a bacteriophage related to bacteriophage phi 11.

A thymine-requiring mutant of Staphylococcus aureus, strain 8325 (PI258)thy, undergoes prophage induction and lysis after thymine starvation. Four different phages were isolated from the lysate in low titers, among which was a phage designated phi 14, which differs from phage phi 11 in its immunity locus. The thymineless induced lysates of strain 8325(PI258)thy transduce the penicillinase plasmid at high frequency (10(-1), whereas transduction of chromosomal markers is inefficient. A plasmic-cured derivative of strain 8325(PI258)thy is also lysed by thymine starvation and be used for high-frequency transduction of other plasmids. Reconstitution of a strain of S. aureus that responds to thymine starvation was only partially successful, but this system can effectively be used to transduce plasmids or plasmid derivatives.     

Isolation of mutants of bacteriophage T4 unable to induce thymidine kinase activity. II. Location of the structural gene for thymidine kinase.

Amber mutants of bacteriophage T4 have been isolated that induce thymidine kinase activity only after infection of a strain of Escherichia coli carrying a suppressor mutation. The activity induced when one of these mutants infected this suppressor strain is much more heat sensitive than the activity induced by wild-type T4. This indicates that this amber mutation lies within the structural gene for thymidine kinase. This gene is between fI and v on the standard T4 genetic map. A mutant of tt4 that is unable to induce thymidine kinase activity incorporates only about one-eighth as much thymidine into its DNA as phage that do induce thymidine kinase. This contrasts to the findings that the total thymidine kinase activity in extracts prepared from cells infected with phage able to induce thymidine kinase in only twice as great as the activity in cells infected with the mutant unable to induce the enzyme.     

l-Pantoyl lactone dehydrogenase from Rhodococcus erythropolis: genetic analyses and application to the stereospecific oxidation of l-pantoyl lactone

The 1,2-propanediol (1,2-PD) inducible membrane-bound L-pantoyl lactone (L-PL) dehydrogenase (LPLDH) has been isolated from Rhodococcus erythropolis AKU2103 (Kataoka et al. in Eur J Biochem 204:799, 1992). Based on the N-terminal amino acid sequence of LPLDH and the highly conserved amino acid sequence in homology search results, the LPLDH gene (lpldh) was cloned. The gene consists of 1,179 bases and encodes a protein of 392 amino acid residues. The deduced amino acid sequence showed high similarity to the proteins of the FMN-dependent α-hydroxy acid dehydrogenase/oxidase family. The overexpression vector pKLPLDH containing lpldh with its upstream region (1,940 bp) was constructed and introduced into R. erythropolis AKU2103. The recombinant R. erythropolis AKU2103 harboring pKLPLDH showed six times higher LPLDH activity than the wild-type strain. Conversion of L-PL to ketopantoyl lactone was achieved with 92% or 80% conversion yield when the substrate concentration was 0.768 or 1.15 M, respectively. Stereoinversion of L-PL to D-PL was also carried out by using the combination of recombinant R. erythropolis AKU2103 harboring pKLPLDH and ketopantoic acid-reducing Escherichia coli.     

Characterization of new regulatory mutants of bacteriophage T4. II. New class of mutants.

Amber mutants of bacteriophage T4 have been isolated that induce thymidine kinase activity only after infection of a strain of Escherichia coli carrying a suppressor mutation. The activity induced when one of these mutants infected this suppressor strain is much more heat sensitive than the activity induced by wild-type T4. This indicates that this amber mutation lies within the structural gene for thymidine kinase. This gene is between fI and v on the standard T4 genetic map. A mutant of tt4 that is unable to induce thymidine kinase activity incorporates only about one-eighth as much thymidine into its DNA as phage that do induce thymidine kinase. This contrasts to the findings that the total thymidine kinase activity in extracts prepared from cells infected with phage able to induce thymidine kinase in only twice as great as the activity in cells infected with the mutant unable to induce the enzyme.     

Transduction of Lactose Metabolism in Streptococcus lactis C2

Ultraviolet (UV)-induced phage lysates, from lactose-positive (lac(+)) Streptococcus lactis C2, transduced lactose fermenting ability to lac(-) recipient cells of this organism. Although the phage titer could not be determined due to the absence of an appropriate indicator strain, the number of transductants was proportional to the amount of phage lysate added. Treatment of the lysate with deoxyribonuclease had no effect on this conversion, indicating the observed genetic change was not mediated by free deoxyribonucleic acid. When the lac(+) transductants were isolated and exposed to UV irradiation, lysates with higher transducing ability were obtained. The transducing ability of this lysate was about 100-fold higher than that observed in the original lysates. The lac(+) transductants were unstable since lac(-) segregants occurred at high frequency. The phage lysate from S. lactis C2 also transduced maltose and mannose metabolism to the respective negative recipient cells. The results demonstrate the transduction of carbohydrate markers by a streptococcal phage and establish a genetic transfer system in group N streptococci.     

Alteration of Host Specificity to Lytic Bacteriophages in Streptococcus cremoris

A mutant of Streptococcus cremoris strain ML1 was isolated based on its resistance to acriflavine. The mutant strain showed resistance to the growth of virulent bacteriophages to which the parental strain was sensitive whereas it became sensitive to a number of other virulent phages to which the parental strain was resistant. At the same time, infection of the mutant strain by another bacteriophage sc607 resulted in killing of cells without production of progeny phages. The phage adsorption appeared normal, suggesting that the killing was a postadsorption event. Such killing of bacterial cells was prevented by chloramphenicol treatment, indicating that involvement of some protein either synthesized by phage or phage-induced cellular protein. Synthesis of ribonucleic acid was abruptly terminated after infection of the mutant strain by phage sc607 but not of the parental strain. The alteration of host specificity in the mutant to different lytic bacteriophages and especially abortive infection by phage sc607 resembles the prophage-mediated interference observed in other bacteria.     

A membrane protein is required for bacteriophage c2 infection of Lactococcus lactis subsp. lactis C2.

Phage-resistant mutants, isolated from cultures of Lactococcus lactis subsp. lactis C2 infected with phage c2, did not form plaques but bound phage normally. The mutants were sensitive to another phage, sk1, although the number of plaques was reduced approximately 56% and the plaques were four times smaller. Binding to phage sk1 was reduced about 10%. Another group of phage-resistant mutants, isolated from cultures infected with phage sk1, bound normally to both phages c2 and sk1 but did not form plaques with either phage. Carbohydrate analyses by gas chromatography of the cell walls showed no significant differences in saccharide compositions between the wild-type and phage-resistant cells. However, a difference was observed in the interactions of the phage with the cytoplasmic membranes. Membranes from the wild-type cells, but not mutant cells, inactivated phage c2. Phage sk1 was not inactivated by membrane from either strain. Treatment of wild-type membranes with proteinase K eliminated the ability of the membrane to inactivate the phage, whereas treatment with mutanolysin had no effect. On the basis of this ability to inactivate the phage, a membrane protein was partially purified by gel filtration and ion-exchange chromatography. Under nondenaturing conditions, the phage-inactivating protein has an apparent Mr of approximately 350,000. The protein has an apparent subunit size of 32 kDa, which suggests that it normally exists as a multimer with 10 to 12 subunits or in association with other membrane components. It is proposed that this protein is required for phage c2 infection.     

Deep desulfurization of diesel oil and crude oils by a newly isolated Rhodococcus erythropolis strain

The soil-isolated strain XP was identified as Rhodococcus erythropolis. R. erythropolis XP could efficiently desulfurize benzonaphthothiophene, a complicated model sulfur compound that exists in crude oil. The desulfurization product of benzonaphthothiophene was identified as alpha-hydroxy-beta-phenyl-naphthalene. Resting cells could desulfurize diesel oil (total organic sulfur, 259 ppm) after hydrodesulfurization. The sulfur content of diesel oil was reduced by 94.5% by using the resting cell biocatalyst for 24 h at 30 degrees C. Biodesulfurization of crude oils was also investigated. After 72 h of treatment at 30 degrees C, 62.3% of the total sulfur content in Fushun crude oil (initial total sulfur content, 3,210 ppm) and 47.2% of that in Sudanese crude oil (initial total sulfur, 1,237 ppm) were removed. Gas chromatography with pulsed-flame photometric detector analysis was used to evaluate the effect of R. erythropolis XP treatment on the sulfur content in Fushun crude oil, and it was shown that most organic sulfur compounds were eliminated after biodesulfurization.