Macromolecular synthesis in mycobacteriophage I3 infected cells

DNA-, RNA- and protein synthesis have been studied inMycobacterium smegmatis cells infected with phage 13. The macromolecular synthesis continued until the end of latent period. Early RNA and protein synthesis were necessary prior to the commencement of DNA replication. The infecting phage DNA sedimented as larger than unit length of genome, after initiation of DNA synthesis. Although the host DNA was not degraded, 90 percent of the RNA synthesized after phage infection hybridized to phage DNA.     

In vivo loss of supercoiled DNA carrying a palindromic sequence

Summary Interest in the fate of long palindromic DNA sequences in E. coli has been kindled by the observation that their inviability is overcome in recBC sbcB strains and that these hosts permit the construction of DNA libraries containing long palindromic sequences present in the human genome. In this paper we show that a reduction in the level of intracellular supercoiled DNA occurs as the result of the presence of a 530 bp palindrome in bacteriophage lambda. This reduction occurs in Rec⁺ and recA strains but not in strains lacking exonucleases V and I (recBC sbcB). However, the DNA must be active (not repressed) for this reduction to be observed, since it is not seen in a Rec⁺ host lysogenic for phage lambda. These results argue against two hypotheses: firstly, that the palindrome causes inviability solely by interfering with packaging, and secondly, that it dose so solely by interfering with recombination. Conversely, these results suggest that a feature of active monomeric DNA (probably its replication) is involved in inviability.     

Transfection of Mycobacterium smegmatis SN2 with mycobacteriophage I3 DNA

Mycobacterium smegmatis SN2 does not exhibit natural competence for the uptake of phage I3 DNA. Competence can artificially be induced by treatment with glycine or CaCl₂, and the combination of both is even more effective. The efficiency of transfection can be improved by inclusion of protamine sulphate and heterologous RNA in the system. From ³²P DNA uptake studies the major barrier for the entry of DNA has been found to be the complex cell wall. The efficiency of transfection calculated on the basis of fraction of DNA which has entered the cell is comparable to that of other bacterial systems. The phage development takes a longer time (7 h for one cycle) after transfection, as compared to infection (4 h).     

Differentiation of metaxylem cell line in the root ofAllium cepa

Summary The first step of differentiation in the root segments ofAllium cepa containing metaxylem cells in different stages of differentiation were studied by DNA reassociation curves and compared to meristem cell extracted DNA. Upon sonication of DNA samples to about 400 base pairs, the reassociation profiles of the heat denatured DNA, were spectrophotometrically followed at two different concentrations. The kinetic complexities,i.e., the number of base pairs per haploid genome of a given sequence and its redundancy were calculated. Differences were found at the level of highly and medium repetitive sequences, thus demonstrating that some DNA reassociation classes may undergo amplification during root development.     

Nucleotide sequence of the genome of the filamentous bacteriophage I2-2: Module evolution of the filamentous phage genome

Summary The nucleotide sequence of the circular single-stranded genome of the filamentous Escherichia coli phage I2-2 has been determined and compared with those of the filamentous E. coli phages Ff(M13, fl, or fd) and IKe. The I2-2 DNA sequence comprises 6744 nucleotides; 139 nucleotides less than that of the N- and I2-plasmid-specific phage IKe, and 337 (336) nucleotides more than that of the F-plasmid-specific phage Ff. Nucleotide sequence comparisons have indicated that I2-2, IKe, and Ff have a similar genetic organization, and that the genomes of I2-2 and IKe are evolutionarily more closely related than those of I2-2 and Ff. The studies have further demonstrated that the I2-2 genome is a composite replicon, composed of only two-thirds of the ancestral genome of IKe. Only a contiguous I2-2 DNA sequence of 4615 nucleotides encompassing not only the coat protein and phage assembly genes, but also the signal required for efficient phage morphogenesis, was found to be significantly homologous to sequences in the genomes of IKe and Ff. No homology was observed between the consecutive DNA sequence that contains the origins for viral and complementary strand replication and the replication genes. Although other explanations cannot be ruled out, our data strongly suggest that the ancestor filamentous phage genome of phages I2-2 and IKe has exchanged its replication module during evolution with that of another replicon, e.g., a plasmid that also replicates via the so-called rolling circle mechanism. Offprint requests to: R.N.H. Konings     

Deoxyribonucleic acid replication time in Mycobacterium tuberculosis H37 Rv

The DNA increment method, designed for measuring the increment in the amount of DNA after inhibition of initiation of fresh rounds of replication initiation was employed to measure the rate of deoxyribonucleic acid (DNA) chain growth in Mycobacterium tuberculosis H37Rv growing in Youman and Karlson's medium at 37°C with a generation time of 24 h and also in relatively fast growing species like Mycobacterium smegmatis and Escherichia coli. From the results obtained, the time required for a DNA replication fork to traverse the chromosome from origin to terminus (C period) was calculated. The chain elongation rates of DNA of the three organisms was determined from the C period and the known genome sizes assuming that all these genomes have a single replication origin and bidirectional replication fork. The rate for M. tuberculosis was 3,200 nucleotides per min about 11 times slower than that of M. smegmatis and about 13–18 times slower than that of E. coli.Abbreviations DNA deoxyribonucleic acid - td delay in initiation - OD optical density - CAM chloramphenicol - RIF rifampicin     

An orphan gyrB in the Mycobacterium smegmatis genome uncovered by comparative genomics

DNA gyrase is an essential topoisomerase found in all bacteria. It is encoded bygyrB andgyrA genes. These genes are organized differently in different bacteria. Direct comparison ofMycobacterium tuberculosis andMycobacterium smegmatis genomes reveals presence of an additionalgyrB inM. smegmatis flanked by novel genes. Analysis of the amino acid sequence of GyrB from different organisms suggests that the orphan GyrB inM. smegmatis may have an important cellular role.     

Production of recombinant proteins in Mycobacterium smegmatis for structural and functional studies

Protein production using recombinant DNA technology has a fundamental impact on our understanding of biology through providing proteins for structural and functional studies. Escherichia coli (E. coli) has been traditionally used as the default expression host to over‐express and purify proteins from many different organisms. E. coli does, however, have known shortcomings for obtaining soluble, properly folded proteins suitable for downstream studies. These shortcomings are even more pronounced for the mycobacterial pathogen Mycobacterium tuberculosis, the bacterium that causes tuberculosis, with typically only one third of proteins expressed in E. coli produced as soluble proteins. Mycobacterium smegmatis (M. smegmatis) is a closely related and non‐pathogenic species that has been successfully used as an expression host for production of proteins from various mycobacterial species. In this review, we describe the early attempts to produce mycobacterial proteins in alternative expression hosts and then focus on available expression systems in M. smegmatis. The advantages of using M. smegmatis as an expression host, its application in structural biology and some practical aspects of protein production are also discussed. M. smegmatis provides an effective expression platform for enhanced understanding of mycobacterial biology and pathogenesis and for developing novel and better therapeutics and diagnostics.     

L5 luciferase reporter mycobacteriophages: a sensitive tool for the detection and assay of live mycobacteria

Recombinant bacteriophages provide efficient delivery systems for introducing reporter genes into specific bacterial hosts. We have constructed mycobacteriophage L5 recombinants carrying the firefly luciferase gene inserted into the tRNA region of the phage genome. Infection of Mycobacterium smegmatis by these phages results in expression of the luciferase gene and light emission. Fortuitously, the luciferase gene is expressed continuously in lysogens surviving infection. Synthesis of luciferase from a mycobacterial promoter created by cloning enables the detection of extremely small numbers of M. smegmatis cells. These reporter phages can be used to discriminate between drug-sensitive and drug-resistant strains of M. smegmatis, and may provide tools for the rapid identification and classification of antimycobacterial agents.     

Physical and functional interactions between 3-methyladenine DNA glycosylase and topoisomerase I in mycobacteria

DNA glycosylases play important roles in DNA repair in a variety of organisms, including humans. However, the function and regulation of these enzymes in the pathogenic bacterium Mycobacterium tuberculosis and related species are poorly understood. In the present study, the physical and functional interactions between 3-methyladenine DNA glycosylase (MAG) and topoisomerase I (TopA) in M. tuberculosis and M. smegmatis were characterized. MAG was found to inhibit the function of TopA in relaxing supercoiled DNA. In contrast, TopA stimulated the cleavage function of MAG on a damaged DNA substrate that contains hypoxanthine. The interaction between the two proteins was conserved between the two mycobacterial species. Several mutations in MAG that led to the loss of its interaction with and activity regulation of TopA were also characterized. The results of this study further elucidate glycosylase regulation in both M. smegmatis and M. tuberculosis.     

Nuclear genome characterization and carrageenan analysis ofGymnogongrus griffithsiae (Rhodophyta) from North Carolina

DNA reassociation kinetics were used to determine nuclear genome organization and complexity inGymnogongrus griffithsiae (Gigartinales, Rhodophyta). Results indicate the presence of three second order components corresponding to fast (3%), intermediate (8%) and slow (89%) fractions. Thus the genome consists mainly of unique sequences. Thermal denaturation (T _m) indicated a nuclear DNA base pair composition of 40 mol% G + C. Microspectrophotometry with the DNA-localizing fluorochrome DAPI was used to confirm ploidy level differences in the gametophytic and tetrasporoblastic phases. Comparisons of mean nuclear DNA (I _f) values to chicken erythrocytes (RBC) resulted in an estimate of 0.32 pg/2 C genome forGymnogongrus griffithsiae. Karyological studies using aceto-orcein revealed the presence of ca. 23 bivalents during diakinesis of tetrasporangial mother cells. Total carrageenan content in water extraction was 30% dry weight. Infrared spectroscopy confirmed the isolated carrageenan to be the iota-fraction.     

Isolation and characterization of new bacteriophages for Myxococcus xanthus

Bacteriophages for Myxococcus xanthus of similar morphology to phage Mx4 were isolated from cultures of a variety of myxobacterial species. Phages similar to Mx1 and Mx8 were obtained by infecting M. xanthus with one of the phages of the Mx4 group that had been treated with either UV light or a chemical mutagen.The DNA molecules from the phages were characterized by electron microscopy. One phage, Mx113, contains an unusual type of terminal redundancy revealed by examination of denatured and re-annealed DNA.Several of the phages of the Mx4 group and the other two new phages, Mx113 and Mx811, were found capable of transducing genetic markers in M. xanthus.One phage, Mx416, was characterized in more detail. It establishes true lysogens in M. xanthus; the phage plaques on both a non-motile mutant and also on a wild-type host although it is restricted in the latter.We dedicate this paper to Professor Dr. Hans Kühlwein in the year of his retirement and in recognition of his many contributions to the study of Myxobacteria     

Metabolism of cyclic AMP in non-pathogenic Mycobacterium smegmatis

The intracellular concentration of cyclic AMP reached a maximum in 3.5-day old cultures of Mycobacterium smegmatis grown in the presence of glycerol as the main source of carbon. Glucose-grown cells exhibited decreased cyclic AMP levels at all stages of growth. When M. smegmatis cells were incubated with various metabolites, pyruvate increased whereas glucose, citric acid, succinic acid and lactic acid decreased intracellular cyclic AMP levels. No cyclic AMP was detected in the incubation medium. The presence of a cyclic AMP-binding protein was demonstrated in cellfree extracts of M. smegmatis.     

一株类志贺邻单胞菌噬菌体生物学特性及全基因组分析

[目的]多重耐药菌株的出现给食品安全带来严重威胁.噬菌体是不同于抗生素的一类重要杀菌因子,对其生物学特性及基因组的研究和分析可为噬菌体的抗菌应用提供依据.[方法]对噬菌体phiP4-7的生物学特性、基因组学、分类学进行研究.[结果]经透射电子显微镜观察,确定phiP4-7头部直径为(50.59±1.68) nm,尾部长...     

Characterization of filamentous bacteriophage PE226 infecting Ralstonia solanacearum strains

Aims: The aim of this study was to isolate and characterize new bacteriophages that infect a wide range of plant pathogenic Ralstonia solanacearum strains. Methods and Results: Fifteen bacteriophages were isolated from pepper, tomato and tobacco plant rhizospheres infected with R. solanacearum. A host specificity analysis of the isolated phages using nine strains of R. solanacearum indicated great phage diversity in a single soil. Two phages, PE226 and TM227, showed clear plaques on all nine bacterial hosts tested and were virtually identical in morphology and genome. PE226, an Inovirus, is a long, flexible, filamentous phage carrying a circular (+) sense single‐strand DNA genome of 5475 nucleotides. DNA sequences of PE226 exhibited nine open reading frames (ORF) that were not highly similar to those of other phages infecting R. solanacearum. The genome organization of PE226 was partially similar to that of p12J of Ralstonia pickettii. One ORF of PE226 showed identity to the zot gene encoding zonula occludens toxin of Vibrio cholera. Orf7 of PE226 was also present in the genome of R. solanacearum strain SL341. However, SL341, a highly virulent strain in tomato, was still sensitive to phage PE226. Conclusions: A new, flexible, filamentous phage PE226 infected wide range of R. solanacearum strains and carried unique circular single‐strand DNA genome with an ORF encoding Zot‐like protein. Significance and Impact of the Study: PE226 may be a new type of temperate phage, based on its lytic nature on a wide range of hosts and the presence of a zot homologue in a host bacterial genome.     

Isolation and characterization of a virulent phage for Rhodobacter sphaeroides

A new virulent bacteriophage, termed øRsV, was isolated from a local sewage plant on the facultative phototrophic bacterium Rhodobacter sphaeroides DSM 159 as the host organism. Electron microscopic studies revealed that in general morphology phage øRsV resembles the T-even Escherichia coli phages. The host range of phage øRsV was restricted to strains of R. sphaeroides. E. coli strains B and K 12 were not infected. The phage genome was characterized on the basis of thermal denaturation profiles and restriction analyses indicating that it consists of about 160 kb of double-stranded DNA lacking cohesive ends. The G+C content was determined to be 46.8 mol%.     

DNA sequence organization in the genome of Nicotiana tabacum

The genome of Nicotiana tabacum was investigated by DNA/DNA reassociation for its spectrum of DNA repetition components and pattern of DNA sequence organization. The reassociation of 300 nucleotide DNA fragments analyzed by hydroxyapatite chromatography reveals the presence of three major classes of DNA differing in reiteration frequency. Each class of DNA was isolated and characterized with respect to kinetic homogeneity and thermal properties on melting. These measurements demonstrate that the genome of N. tabacum has a 1C DNA content of 1.65 pg and that DNA sequences are represented an average of 12,400, 252, and 1 times each. — The organization of the DNA sequences in the N. tabacum genome was determined from the reassociation kinetics of long DNA fragments as well as S1 nuclease resistance and hyperchromicity measurements on DNA fragments after annealing to C₀t values at which only repetitive DNA sequences will reassociate. At least 55% of the total DNA sequences are organized in a short period interspersion pattern consisting of an alternation of single copy sequences, averaging 1400 nucleotides, with short repetitive elements approximately 300 nucleotides in length. Another 25% of the genome contains long repetitive DNA sequences having a minimal genomic length of 1500 nucleotides. These repetitive DNA sequences are much less divergent than the short interspersed DNA sequence elements. These results indicate that the pattern of DNA sequence organization in the tobacco genome bears remarkable similarity to that found in the genomes of most animal species investigated to date.     

诺卡氏菌烈性噬菌体vB_Ncarnea_KYD1的分离纯化与基因组分析

【背景】诺卡氏菌是一种广泛分布的好氧放线菌,可在人体内引起局部或播散性感染,尤其是在免疫功能低下的个体中。诺卡氏菌感染在临床上较难鉴定,而且不断有新型诺卡氏菌种被发现。不同类型、不同地域的诺卡氏菌具有流行差异和抗生素敏感性差异,阻碍了适当治疗方式的选择。利用病灶处的宿主菌分离得到噬菌体来控制诺卡氏菌感染的这种方法在近年来受到了各界的关注。【目的】尝试从环境中分离出能够用于临床治疗的针对诺卡氏菌的烈性噬菌体,并研究其基因组学特征。【方法】利用双层平板法分离得到目标噬菌体,观察其噬菌斑形态,并对噬菌体进行分离纯化,在透射电镜下鉴定其特征。提取噬菌体DNA进行全基因组测序与注释,并与数据库内已知噬菌体基因组进行比较,同时构建系统进化树以进行遗传进化分析。【结果】本文以肉色诺卡氏菌为宿主,从环境样本中分离出一株烈性噬菌体vB_Ncarnea_KYD1,在双层平板上可形成直径<2 mm的透亮均匀的噬菌斑。基因组分析表明,vB_Ncarnea_KYD1DNA为环状,大小为66 621 bp,共发现102个蛋白质编码区(coding sequence,CDS)及一个tRNA-Ser编码序列。透射电镜观察与系统进化树综合分析可以确定,vB_Ncarnea_KYD1为长尾噬菌体科的一个新属。其在进化过程中经历了复杂的基因重组过程。暂未发现毒力因子相关基因与抗性基因,具备实用价值。【结论】从环境水体中分离出一株烈性肉色诺卡氏菌噬菌体vB_Ncarnea_KYD1,通过电镜观察与基因组分析可知,此株噬菌体为长尾噬菌体,基因组中暂未发现不利于临床应用的相关基因,是一株相对安全的烈性诺卡氏菌噬菌体。研究结果丰富了国内噬菌体资源库,并为后续诺卡氏菌感染疾病的治疗提供支持。