Searching for a Bacteriophage Lysin to Treat Corynebacterium bovis in Immunocompromised Mice.

Corynebacterium bovis is the causative agent of Corynebacterium-associated hyperkeratosis in immunocompromised mice. The resulting skin pathology can be profound and can be associated with severe wasting, making the animals unsuitable for research. Although the administration of antibiotics is effective in resolving clinical symptoms, antibiotics do not eradicate the offending bacterium. Furthermore, antibiotic use may be contraindicated as it can affect tumor growth and is associated with Clostridioides difficile enterotoxemia in highly immunocompromised murine strains. Lysins, which are lytic enzymes obtained from bacteriophages, are novel antimicrobial agents for treating bacterial diseases. The advantage of lysins are its target specificity, with minimal off-target complications that could affect the host or the biology of the engrafted tumor. The aim of this study was to identify lysins active against C. bovis. Chemical activation of latent prophages by using mitomycin C in 3 C. bovis isolates did not cause bacteriophage induction as determined through plaque assays and transmission electron microscopy. As an alternative approach, 8 lysins associated with other bacterial species, including those from the closely related species C. falsenii, were tested for their lytic action against C. bovis but were unsuccessful. These findings were congruent with the previously reported genomic analysis of 21 C. bovis isolates, which failed to reveal bacteriophage sequences by using the PHAST and PHASTER web server tools. From these results, we suggest C. bovis is among those rare bacterial species devoid of lysogenic bacteriophages, thus making the identification of C. bovis-specific lysins more challenging. However, C. bovis may be a useful model organism for studying the effects of antiphage systems.

Complete genome analysis of a novel temperate bacteriophage induced from Corynebacterium striatum.

A temperate bacteriophage, IME1320_01, was induced by mitomycin C treatment from Corynebacterium striatum. This phage possesses a double-stranded DNA genome of 40,086 bp with a G+C content of 58%. A total of 53 putative open reading frames (ORFs) were identified in its genome. BLASTn analysis revealed that IME1320_01 had the highest sequence similarity to Corynebacterium striatum strain 216, with a genome homology coverage of 44% and highest sequence identity of 95%. The termini of the phage genome was non-redundant, with a 13-nt 3'-protruding cohesive end. To the best of our knowledge, phage IME1320_01 is the first inducible phage to be identified in Corynebacterium striatum.

Resolving the Complexity of Human Skin Metagenomes Using Single-Molecule Sequencing.

UNLABELLED: Deep metagenomic shotgun sequencing has emerged as a powerful tool to interrogate composition and function of complex microbial communities. Computational approaches to assemble genome fragments have been demonstrated to be an effective tool for de novo reconstruction of genomes from these communities. However, the resultant "genomes" are typically fragmented and incomplete due to the limited ability of short-read sequence data to assemble complex or low-coverage regions. Here, we use single-molecule, real-time (SMRT) sequencing to reconstruct a high-quality, closed genome of a previously uncharacterized Corynebacterium simulans and its companion bacteriophage from a skin metagenomic sample. Considerable improvement in assembly quality occurs in hybrid approaches incorporating short-read data, with even relatively small amounts of long-read data being sufficient to improve metagenome reconstruction. Using short-read data to evaluate strain variation of this C. simulans in its skin community at single-nucleotide resolution, we observed a dominant C. simulans strain with moderate allelic heterozygosity throughout the population. We demonstrate the utility of SMRT sequencing and hybrid approaches in metagenome quantitation, reconstruction, and annotation. IMPORTANCE: The species comprising a microbial community are often difficult to deconvolute due to technical limitations inherent to most short-read sequencing technologies. Here, we leverage new advances in sequencing technology, single-molecule sequencing, to significantly improve reconstruction of a complex human skin microbial community. With this long-read technology, we were able to reconstruct and annotate a closed, high-quality genome of a previously uncharacterized skin species. We demonstrate that hybrid approaches with short-read technology are sufficiently powerful to reconstruct even single-nucleotide polymorphism level variation of species in this a community.

Aspectos clínico-epidemiológicos e microbiológicos de processos infecciosos causados por Corynebacterium spp em pacientes de centro de referência oncológico – Rio de Janeiro, Brasil / Clinical-epidemiological and microbiological aspects of infectious processes caused by Corynebacterium spp in pacients at the National Cancer Institute in Rio de Janeiro, Brazil

Os resultados permitiram a redação de quatro artigos. Aspectos microbiológicos e clínicos de corinebacterioses em pacientes com câncer observados durante cinco anos foram descritos no Artigo 1. No Artigo 2 foram apresentados casos de bacteremia causados por corinebactérias invasivas não toxigênicas em dois períodos com intervalo de sete anos. As infecções em pacientes com câncer por C. diphtheriae, causando casos clínicos atípicos foram descritas no Artigo 3, além do estudo dos principais fatores de virulência de uma cepa de C. diphtheriae isolada de infecção associada ao cateter de nefrostomia foi descrita no Artigo 4. Resumidamente no Artigo 1, além dos aspectos clínico-epidemiológicos foram avaliados os perfis de resistência aos antimicrobianos e o potencial de virulência dos micro-organismos. Em cinco anos, 932 amostras de corinebactérias, com perfis de resistência aos antimicrobianos testados, foram isoladas de pacientes com câncer. As espécies predominantes foram Corynebacterium amycolatum (44,7%), Corynebacterium minutissimum (18,3%) e Corynebacterium pseudodiphtheriticum (8,5%). O uso de catéteres de longa permanência e a neutropenia, foram às condições importantes para infecção por corinebactérias. As doenças de base mais comuns foram os tumores sólidos. Pacientes hospitalizados apresentaram risco seis vezes maior de morrer, quando relacionadas às taxas de mortalidade com 30 dias (RC= 5,5; IC 95%= 1,15-26,30; p= 0,033). As bacteremias (Artigo 2) causadas por corinebactérias foram observadas em dois períodos: 2003-2004 (n=38) e de 2012-2013 (n=24). As espécies multirresistentes C. amycolatum e Corynebacterium jeikeium foram os principais responsáveis pelos quadros de bacteremia. Havia 34 pacientes com tumores sólidos e 28 pacientes com doenças linfoproliferativas, sendo que 21 deles apresentavam neutropenia e 54 utilizavam cateter venoso central. Em 41 pacientes havia infecção relacionada ou associada aos dispositivos intravasculares...

A retrospective study at the InstitutoNacional doCâncer-INCA (National Cancer Institute) in Rio de Janeiro, Brazil examined infections of Corynebacterium sp. in cancer patients. The results were presented in four papers. Article 1 describes microbiological and clinical aspects of corynebacteriosis in cancer patients observed over five years. Article 2 presents cases of bacteremia caused by invasive non-toxigenic corynebacteria observed in 2003-2004 and seven years later in 2012-2013. Article 3 presents atypical clinical cases of cancer patients infected by Corynebacterium diphtheriae, while Article 4 is a study of the major bacterial virulence factors of an isolated strain of C. diphtheriae in infections associated with nephrostomy catheters.In addition to clinical and epidemiological aspects, Article 1 evaluates the antimicrobial resistance profiles and potential virulence factor of microorganisms. Over a period of five years, 932 samples of corynebacteria with antimicrobial resistance profiles were isolated from patients with cancer. The predominant species were Corynebacterium amycolatum (44.7%), Corynebacterium minutissimum (18.3%) and Corynebacterium pseudodiphtheriticum (8.5%).Long-term catheter use and neutropenia were the major conditions for infection by corynebacteria. Solid tumors were the most common underlying illness. The 30-day mortality rate for patients with corynebacteria infections was six times greater in hospitalized patients than for non-hospitalized patients (OR = 5.5, 95% CI = 1.15 to 26.30, p = 0.033).In Article 2, bacteremia caused by corynebacteria were observed in two time frames: 2003 to 2004 (n=38) and 2012 to 2103 (n=24). The multidrug-resistant species C. amycolatum and Corynebacterium jeikeium were responsible for invasive diseases.There were 34 patients with solid tumors and 28 patients with lymphoproliferative diseases, of which 21 had neutropenia and 54 used central venous catheter...

Effects of environmental modification on mastitis occurrence and hormonal changes in Holstein cows / Efeito da modificação ambiental sobre a ocorrência de mastite e alterações hormonais de vacas Holandesas

The purpose of this research was to evaluate the effects of evaporative cooling in freestall on mastitis occurrence, milk production, and composition, as well as cortisol, T3 (triiodothyronine), and T4 (thyroxin) levels in lactating dairy cows. Twenty-eight multiparous cows averaging 70 ± 10 day postpartum were used in four treatments from January to March 2003. The treatments were: Day (cooling from 7:00 a.m. to 7:00 p.m.); Night (cooling from 7:00 p.m. to 7:00 a.m.); 24-hour (cooling 24-hour); and Control (no cooling). Wired cup test was used for clinical mastitis diagnosis, and the California Mastitis Test (CMT) was used to identify subclinical mastitis. Blood and milk samples were taken weekly for microbiological and hormonal analyses. The cortisol levels were higher than normal values in all treatment groups, suggesting stress conditions, but T3 and T4 levels remained normal in all groups. The occurrence of subclinical mastitis was lower in Day and Night groups than in Control and 24-hour groups. Regarding the microbiological analyses, in all groups the isolation of Corynebacterium sp. from milk samples increased while negative coagulase staphylococci (CNS) declined as etiological agents of subclinical mastitis. However, in Day and 24-hour groups, coagulase positive staphylococci (CPS) increased mainly Staphylococcus aureus (49.8% and 47.7% respectively). The Night group showed a decrease in subclinical mastitis occurrences. Our data indicate that all animals subjected to treatments presented high levels of cortisol, indicating a stress condition. The Night treatment presented a reduction in microbial isolation, suggesting a reduced susceptibility to mastitis.

O trabalho teve como objetivo avaliar a eficiência do sistema de resfriamento adiabático evaporativo, acionado em diferentes horários, em instalação do tipo freestall e seus reflexos sobre a ocorrência de mastite, produção e composição do leite e respostas hormonais de vacas em lactação. Foram utilizadas 28 vacas em lactação (70±10 dias), multíparas, das raças Holandesa Preta e Branca e Pardo Suíça, com produção média diária de 23±2,3 kg leite/dia. O período experimental de 56 dias teve início em 20 de janeiro de 2003. Os tratamentos foram: Controle (sem resfriamento); Dia (resfriamento 7 as 19 h); Noite (resfriamento 19 às 7 h) e 24 horas (resfriamento durante 24 h). A temperatura de bulbo seco (TBS), umidade relativa do ar (UR) e a temperatura de globo negro (TGN) foram mensuradas ao longo das 24 horas. A ordenha foi realizada às 7 h e 19 h. Amostragens semanais de leite e sangue foram realizadas para análise da composição do leite (gordura, proteína, lactose e contagem de células somáticas) e determinações hormonais de cortisol, tiroxina (T4) e triiodotironina (T3). Para avaliação da ocorrência de mastite clínica e subclínica foram feitos exames semanais de TAMIS (caneca de fundo preto) e California Mastitis Test (CMT). Foram colhidas amostras de leite de todos os quartos para identificação microbiológica dos agentes causais da mastite. O tratamento Dia diminuiu (P<0,05) a temperatura do freestall em 5,3°C às 12h e em 3,5°C às 14h em relação ao grupo Controle. A umidade relativa esteve elevada (P<0,05) às 7h no tratamento Noite e às 12h, 14h e 21h no tratamento Dia. Os maiores valores de ITU foram registrados no tratamento Noite às 12h, 14h e 21h. Não foram observadas diferenças entre os tratamentos (P>0,05) para a produção e composição do leite. Nos animais do tratamento Os níveis de cortisol mostraram-se acima (P<0,05) dos níveis normais em todos os tratamentos. Já os teores de T3 e T4 estiveram dentro da faixa de normalidade. Na fase pré-experimental a maior frequência de isolamento bacteriano foi para Staphylococcus coagulase negativa. No tratamento noite e dia, houve uma diminuição na proporção de casos positivos de mastite subclínica da fase pré-experimental em relação à última semana da fase experimental. Na última semana da fase experimental houve uma diminuição de Staphylococcus coagulase negativa e aumento da ocorrência de Corynebacterium sp.

Corynebacterium ulcerans 0102 carries the gene encoding diphtheria toxin on a prophage different from the C. diphtheriae NCTC 13129 prophage.

BACKGROUND: Corynebacterium ulcerans can cause a diphtheria-like illness, especially when the bacterium is lysogenized with a tox gene-carrying bacteriophage that produces diphtheria toxin. Acquisition of toxigenicity upon phage lysogenization is a common feature of C. ulcerans and C. diphtheriae. However, because of a lack of C. ulcerans genome information, a detailed comparison of prophages has not been possible between these two clinically important and closely related bacterial species. RESULTS: We determined the whole genome sequence of the toxigenic C. ulcerans 0102 isolated in Japan. The genomic sequence showed a striking similarity with that of Corynebacterium pseudotuberculosis and, to a lesser extent, with that of C. diphtheriae. The 0102 genome contained three distinct prophages. One of these, ΦCULC0102-I, was a tox-positive prophage containing genes in the same structural order as for tox-positive C. diphtheriae prophages. However, the primary structures of the individual genes involved in the phage machinery showed little homology between the two counterparts. CONCLUSION: Taken together, these results suggest that the tox-positive prophage in this strain of C. ulcerans has a distinct origin from that of C. diphtheriae NCTC 13129.

Identification of a bifunctional primase-polymerase domain of corynephage BFK20 replication protein gp43.

Replication protein gp43 is a gene product of orf43, from the genome of corynephage BFK20 and carries two different domains. The C-terminal part of gp43 is similar to F4-type helicases and the N-terminal part resembles the rare primase-polymerase (prim-pol) domain. We expressed the 372 amino acids of the gp43 N-terminus in the pET expression system as recombinant protein gp43N with His-Tag fusion on both the N- and C-termini. The protein gp43N was purified by immobilized cobalt or nickel ion affinity chromatography. Gel filtration chromatography on Superose 12 showed that the purified protein elutes at an apparent molecular weight of 80 kDa, suggesting that it may be a dimer. We detected primase and DNA polymerase activities in gp43N using a simple method based on the determination of inorganic pyrophosphate and we demonstrated these two activities by polyacrylamide and agarose gel electrophoresis. In both primase and polymerase reactions, gp43N used only deoxyribonucleotides. By using defined single-stranded oligonucleotides as templates, we found that the primase is not highly sequence specific and does not require a specific trinucleotide for initiation of primer synthesis. The prim-pol domain of gp43 is the first such domain of a phage protein studied as an individual heterologous protein.

Endolysin of bacteriophage BFK20: evidence of a catalytic and a cell wall binding domain.

A gene product of ORF24' was identified on the genome of corynephage BFK20 as a putative phage endolysin. The protein of endolysin BFK20 (gp24') has a modular structure consisting of an N-terminal amidase_2 domain (gp24CD) and a C-terminal cell wall binding domain (gp24BD). The C-terminal domain is unrelated to any of the known cell wall binding domains of phage endolysins. The whole endolysin gene and the sequences of its N-terminal and C-terminal domains were cloned; proteins were expressed in Escherichia coli and purified to homogeneity. The lytic activities of endolysin and its catalytic domain were demonstrated on corynebacteria and bacillus substrates. The binding activity of cell wall binding domain alone and in fusion with green fluorescent protein (gp24BD-GFP) were shown by specific binding assays to the cell surface of BFK20 host Brevibacterium flavum CCM 251 as well as those of other corynebacteria.

[Phage resistance of Corynebacterium crenatum conferred by the restriction and modification system cglI].

In order to prevent phage contamination in amino acid fermentation, we introduced the restriction and modification system cglI gene complex into Corynebacterium crenatum and studied their phage-resistance. The cglI gene complex was amplified from Corynebacterium glutamicum by PCR and constructed into pJL23 vector. The recombinant strains were obtained by transformation of the recombinant plasmid pJL23-cglI into C. crenatum. Results showed that the recombinant strains possessed strong phage-resistance activity and broad phage-resistance spectrum, demonstrating the feasibility of using cglI gene complex for construction of phage-resistance recombinant C. crenatum strains and presenting a powerful way to solve the problem of phage contamination in amino acid fermentation industry.

Properties of corynephage attachment site and molecular epidemiology of Corynebacterium ulcerans isolated from humans and animals in Japan.

Sporadic reports of Corynebacterium ulcerans infection in humans and animals have become increasingly common throughout the world. Between 2001 and 2006, five human cases, in addition to isolation of the bacterium from the carcasses of Orcinus orca and Panthera leo, were reported in Japan. While an isolate from P. leo generated only phospholipase D (PLD), the other isolates produced both PLD and diphtheria-like toxin (DLT). Pulsed-field gel electrophoresis analysis showed that isolates from P. leo and humans were genetically homologous. Southern blotting found that a human isolate was lysogenized by two corynephages coding DLT. Sequence analysis of the region of the DLT gene revealed that the integration in C. ulcerans occurred in the same manner as that in C. diphtheriae.

Bacteriophage-based vectors for site-specific insertion of DNA in the chromosome of Corynebacteria.

In Corynebacterium diphtheriae, diphtheria toxin is encoded by the tox gene of some temperate corynephages such as beta. beta-like corynephages are capable of inserting into the C. diphtheriae chromosome at two specific sites, attB1 and attB2. Transcription of the phage-encoded tox gene, and many chromosomally encoded genes, is regulated by the DtxR protein in response to Fe(2+) levels. Characterizing DtxR-dependent gene regulation is pivotal in understanding diphtheria pathogenesis and mechanisms of iron-dependent gene expression; although this has been hampered by a lack of molecular genetic tools in C. diphtheriae and related Coryneform species. To expand the systems for genetic manipulation of C. diphtheriae, we constructed plasmid vectors capable of integrating into the chromosome. These plasmids contain the beta-encoded attP site and the DIP0182 integrase gene of C. diphtheriae NCTC13129. When these vectors were delivered to the cytoplasm of non-lysogenic C. diphtheriae, they integrated into either the attB1 or attB2 sites with comparable frequency. Lysogens were also transformed with these vectors, by virtue of the second attB site. An integrated vector carrying an intact dtxR gene complemented the mutant phenotypes of a C. diphtheriae DeltadtxR strain. Additionally, strains of beta-susceptible C. ulcerans, and C. glutamicum, a species non-permissive for beta, were each transformed with these vectors. This work significantly extends the tools available for targeted transformation of both pathogenic and non-pathogenic Corynebacterium species.

Complete nucleotide sequence and genome analysis of bacteriophage BFK20--a lytic phage of the industrial producer Brevibacterium flavum.

The entire double-stranded DNA genome of bacteriophage BFK20, a lytic phage of the Brevibacterium flavum CCM 251--industrial producer of L-lysine--was sequenced and analyzed. It consists of 42,968 base pairs with an overall molar G + C content of 56.2%. Fifty-five potential open reading frames were identified and annotated using various bioinformatics tools. Clusters of functionally related putative genes were defined (structural, lytic, replication and regulatory). To verify the annotation of structural proteins, they were resolved by 2D gel electrophoresis and were submitted to N-terminal amino acid sequencing. Structural proteins identified included the portal and major and minor tail proteins. Based on the overall genome sequence comparison, similarities with other known bacteriophage genomes include primarily bacteriophages from Mycobacterium spp. and some regions of Corynebacterium spp. genomes--possible prophages. Our results support the theory that phage genomes are mosaics with respect to each other.

Resistance of corynebacterial strains to infection and lysis by corynephage BFK 20.

AIMS: Defence mechanisms of the corynebacterial strains against corynephage BFK 20, which causes lysis of Brevibacterium flavum CCM 251. METHODS AND RESULTS: We tested adsorption of the phage BFK 20 to the corynebacterial cell surface. We observed strong adsorption ranging from ca 79 to 93% on the cells of B. flavum ATCC strains, but only ca 76% for B. flavum CCM 251. Minor adsorption for Brevibacterium lactofermentum BLOB (ca 13%) and no adsorption for Corynebacterium glutamicum RM3 were determined. BFK 20 infection had no significant effect on growth and viability of C. glutamicum and B. lactofermentum, but significantly influenced growth and viability of B. flavum ATCC 21127, 21128 and 21474. Cell growth stopped in short time after infection but with no lysis. Brevibacterium flavum CCM 251 cell growth was arrested too and lysis occurred. The Southern hybridization confirmed the presence of significant amount of BFK 20 DNA in samples from B. flavum CCM 251 and B. flavum ATCC strains after BFK 20 infection. Only weak hybridization signal was detected for DNA from infected cells of B. lactofermentum BLOB and no signal for C. glutamicum RM3. CONCLUSIONS: Based on the above results we suggest presence of a mechanism leading to abortive infection in B. flavum ATCC 21127, 21128 and 21474. In B. lactofermentum BLOB and C. glutamicum RM3 the adsorption barrier is more likely. SIGNIFICANCE AND IMPACT OF THE STUDY: This study increases the knowledge on defence mechanisms of corynebacteria against bacteriophages.

Fractionation of Corynebacterium pekinense AS 1.299 phage subtypes by anion-exchange chromatography.

A novel method to fractionate phage into its subtypes while fully retaining biological function is reported. Corynebacterium pekinense AS 1.299 phage samples, purified by either conventional ultracentrifugation or gel chromatography on a Superose(R) 6 Prep column (0.78 x 30 cm), were fractionated further into four fractions by anion-exchange chromatography on a Toyopearl SuperQ 650C column (0.5 x 20 cm) with a linear gradient of NaCl concentration from 0.2 to 1.0 M in 0.02 M carbonate-biocarbonate buffer, pH 10.0. Two peaks were identified to be C. pekinense AS 1.299 phages by their ability to infect the host bacteria when inoculated into the culture media, and when examined by electron microscopy. These two types of the phage were found to be morphologically the same except for the difference in the length of their non-contractile tails. Both possessed an isometric head with a diameter of 50 +/- 3 nm, while their tails were 170 +/- 10 and 210 +/- 10 nm, respectively. This simple technique provides a convenient method for phage isolation not only to its species homogeneity, but also to determine its subtype or variant homogeneity.

Site-specific integration of corynephage phi16: construction of an integration vector.

Phi16, a temperate phage induced from Corynebacterium glutamicum ATCC 21792, lysogenizes its host via site-specific recombination. The phage attachment site, attP, was located to a 6.5 kb BamHI fragment of the phi16 genome. This fragment also contained phi16 integrative functions. The minimal phage DNA fragment required for integration was defined. This 1630 bp region contained a large open reading frame, int, encoding a protein of 416 amino acids with similarity in its carboxyl-terminal domain to tyrosine recombinases and particularly to the Xer recombinases. The comparison of the nucleotide sequences of attB, attL, attR, and attP identified a common 29 bp sequence, the core sequence. It lies 11 bp downstream of the 3' end of the integrase gene. phi16 integrase was shown to catalyse site-specific integration in trans to attP with an efficiency of 5x10(3) integrants per microg DNA. The integrating fragment catalysed integration in several Corynebacterium strains that are not infected by phi16, thus enlarging the host spectrum of integrating vectors derived from phi16. In these strains, the phi16 attB site was located in a conserved intergenic region and lies downstream of a clp gene.

Extended host range of a beta-related corynebacteriophage.

Unlike most beta-related phages isolated from Corynebacterium diphtheriae, phage 782 readily plaqued on strains of C. ulcerans and C. pseudotuberculosis. The extended host range of phage 782 was not, however, due to a greater ability of the phage to absorb to bacterial surfaces. Using chemical mutagenesis, a number of phage mutants were isolated which had diminished capacities to infect C. ulcerans, suggesting the existence of a locus (ehr) for extended host range. Pre-lysogenization of C. ulcerans strains with phage 782, but not its mutant form or other beta-related phages, rendered them susceptible to infection by previously excluded phages. An examination of recombinant between phages 782, pie, and beta localized ehr to a 7 kilobase region of DNA including attP. The data are compatible with the notion that ehr encodes an anti-restriction function.

Prophage distribution in coryneform bacteria.

Four temperate bacteriophages of corynebacteria were isolated after UV induction. Phages phi 304L and phi 304S were both induced from Corynebacterium glutamicum ATCC 13058, ATCC 21488, ATCC 21649 and ATCC 21650 strains, and have no known sensitive host. Phages phi 15 and phi 16 were both induced from ATCC 14020 and ATCC 21792. Phage phi 15 formed turbid plaques on Corynebacterium sp. ATCC 21857 and on C. glutamicum ATCC 13058, ATCC 21488, ATCC 21649 and ATCC 21650. Phage phi 16 produced turbid plaques only on C. glutamicum ATCC 21792 cured of prophage phi 16. All these phages belong to the Siphoviridae family. Their genomes consist of a double-stranded DNA with cohesive ends and share no homology with each other. Prophages phi 16, phi 304L and phi 304S were integrated into their respective host chromosomes, whereas prophage phi 15 seemed to persist free in the cell. Cross-hybridizations between phage DNAs and total cellular DNA obtained from 20 strains belonging to the genera Corynebacterium and Brevibacterium did not show the presence of these prophages in strains other than their respective hosts.

Characterization and sequence analysis of the F2 promoter from corynephage BFK20.

F2 promoter from corynephage BFK20 was isolated and characterized. It was functional in Escherichia coli and Corynebacterium glutamicum. Cloning of the F2 promoter into the pJUP05 promoter probe vector caused an increase of the neomycin phosphotransferase II specific activity. According to the Northern blot hybridization the nptII gene was expressed from the cloned F2 promoter. The apparent transcription start point in E. coli and C. glutamicum was determined. The -35 region of F2 promoter showed high similarity to that of E. coli promoter consensus sequence, but its -10 region was G+C rich and had no significant homology to that.