Temperate bacteriophage ZF40 of Erwinia carotovora: phage particle structure and DNA restriction analysis

Structural organization of the temperate bacteriophage ZF40 of Erwinia carotovora was studied. Phage ZF40 proved to be a typical member of the Myoviridae family (morphotype A1). Phage particles consist of an isometric head 58.3 nm in diameter and a contractile 86.3-nm-long tail with a complex basal plate and short tail fibers (31.5 nm). Phage tail sheath, a truncated cone in shape, is characterized by specific packaging of structural subunits. The ZF40 phage genome is 45.8 kb in size, as determined by restriction analysis, and contains DNA cohesive ends. The ZF40 phage of Erwinia carotovora is assumed to be a new species of bacteriophages specific for enterobacteria.     

Defective Lysogeny in Erwinia carotovora

The electron microscopic study of several Erwinia carotovora strains showed that the SOS-induced cells of this pectolytic phytopathogenic bacterium produce particular phage parts (tails, heads, and baseplates) but do not assemble them into fully functional phage particles. E. carotovora cells produced several times greater amounts of phage tails in response to induction by mitomycin C than in response to induction by nalidixic acid. The tails were 128–192 nm in length and 13–21 nm in diameter. Phage heads were characterized by four discrete ranges of diameters: 18, 55–59, 66–75, and 92–98 nm. The diameters of phage baseplates varied from 39 to 53 nm, depending on the particular strain. It was shown that cells of the same species may contain several different types of phage tails and heads. The structural organization of phage tails and baseplates in the nalidixic acid–induced lysate of E. carotovora J2 was studied in more detail. The data obtained suggest that pectolytic phytopathogenic erwinia are characterized by defective polylysogeny.     

A Study of Erwinia carotovora Phage Resistance with the Use of Temperate Bacteriophage ZF40

The causes of the unique phage resistance of the pectinolytic phytopathogenic strains of Erwinia carotovora were studied with the use of temperate bacteriophage ZF40. It was shown that, in these bacteria, the bacteriophage–cell interaction can be substantially blocked at the adsorption level. An adequate indicator for studying the temperate bacteriophages of erwinias was developed on the basis of mutants resistant to macromolecular bacteriocins. Various restriction–modification systems, which influence cell resistance to bacteriophages, were revealed for the first time in E. carotovora. The phage resistance was shown to be determined by the wide occurrence of homoimmune temperate viruses in pectinolytic erwinias.     

Morphological study of five bacteriophages of yellow-pigmented enterobacteria

Two bacteriophages isolated onEnterobacter cloacae (C2, C2F) and three isolated onErwinia herbicola (E3, E16P, E16B) were purified by D₂O gradient centrifugation. Phage-containing fractions were negatively stained and examined by electron microscopy. Phages C2, C2F, E3, and E16P showed an elongated head 153×51 nm and a short noncontractile tail 12 nm long terminated by at least two short fibers. These phages correspond to the rate taxonomic group C3. Big capsomeres composing the phage head were evidenced when phage suspensions in D₂O were stained. Phage E16B showed an elongated head 97×40.5 nm, and a contractile tail 89 nm long. This phage corresponds to the extremely rate group A3.     

The In Vitro Self-Assembly of Supramolecular Structures after the Spontaneous Disassembly of Carotovoricins

The self-assembly of supramolecular structures (empty sheaths and polysheaths of the macromolecular Erwinia carotovora bacteriocins) was studied by electron microscopy in the course of 1- to 2-year incubation of phage particles at 4°C. This study showed that the empty sheaths and polysheaths of the bacteriocins of eight E. carotovora strains spontaneously assemble at the self-assembly centers (or crystallization centers), which have a diameter of 26–65 nm and contain a dense proteinaceous material. The self-assembly center consists of two components, a primer and the structural protein of contracted sheaths. Empty sheaths assembled in the crystallization centers are polar structures synthesized through the stepwise head-to-tail polymerization of monomeric units. The supramolecular structures of two E. carotovora 62A bacteriocins are assembled in a different way. At the early stages of their self-assembly, a reticular structure is formed, which then transforms into very long polysheaths composed of monomers. Along with polysheaths, rounded or lamplike structures 33–117 nm in size composed of the subunits of contracted sheaths are produced. Carotovoricins may serve as suitable objects for the study of the self-assembly of elementary biological structures.     

Exploitation of a new flagellatropic phage of Erwinia for positive selection of bacterial mutants attenuated in plant virulence: towards phage therapy

Aims: To isolate and characterize novel bacteriophages for the phytopathogen, Erwinia carotovora ssp. atroseptica (Eca), and to isolate phage‐resistant mutants attenuated in virulence. Methods and Results: A novel flagellatropic phage was isolated on the potato‐rotting bacterial species, Eca, and characterized using electron microscopy and restriction analysis. The phage, named ΦAT1, has an icosahedral head and a long, contractile tail; it belongs to the Myoviridae family. Partial sequencing revealed the presence of genes with homology to those of coliphages T4, T7 and Mu. Phage‐resistant transposon mutants of Eca were isolated and studied in vitro for a number of virulence‐related phenotypes; only motility was found to be affected. In vivo tuber rotting assays showed that these mutants were attenuated in virulence, presumably because the infection is unable to spread from the initial site of inoculation. Conclusions: The Eca flagellum can act as a receptor for ΦAT1 infection, and resistant mutants are enriched for motility and virulence defects. Significance and Impact of the Study: ΦAT1 is the first reported flagellatropic phage found to infect Eca and has enabled further study of the virulence of this economically important phytopathogen.     

Occurrence of bacteriophage T4 receptor in Erwinia carotovora

Summary We have tested for the presence of the receptor for the Escherichia coli phage T4 in different isolates of the plant pathogenic enterobacteria Erwinia carotora subsp. carotovora and subsp. atroseptica. Several of the isolates appeared to contain a functional T4 receptor as shown by phage adsorption and phage-induced lysis of the bacteria. Two of the isolates could even sustain lytic growth of T4. In addition, we show that the transducing derivative of T4, T4GT7, can be employed to transfer plasmids from E. coli to E. carotovora thus opening up new possibilities for genetic analysis of Erwinia.     

Nucleotide Sequences and Organization of the Genes for Carotovoricin (Ctv) from Erwinia carotovora Indicate That Ctv Evolved from the Same Ancestor as Salmonella typhi Prophage

Carotovoricin Er (CtvEr), which is produced by a plant soft rot disease causative agent, Erwinia carotovora subsp. carotovora Er, is a high-molecular-weight bacteriocin showing Myoviridae phage-tail-like morphology with contractile sheath and plural tail fibers. We determined the complete nucleotide sequences of CtvEr genes on the E. carotovora Er chromosome and report that CtvEr genes consist of lysis cassette, major and minor structural protein gene clusters. Four promoters were identified. The lysis gene cassette, which is composed of the genes for lysis enzyme and holin, was also identified and characterized. The nucleotide sequences and organization of the genes for CtvCGE, which is produced by E. carotovora strain CGE234-M403 with the morphology similar to CtvEr, were also determined and compared to that of CtvEr, and it was found that CtvCGE is almost identical to CtvEr except for tail fibers which are involved in the killing spectra of both bacteriocins. We also explain that the gene organization and the deduced amino acid sequences of both carotovoricins are very close to those of prophage, which is lysogenized in the chromosome on Salmonella enterica serovar Typhi CT18. These findings strongly suggest that Ctv evolved as a phage tail-like bacteriocin from a common ancestor with Salmonella typhi prophage.     

OCCURRENCE OF A TEMPERATE CYANOPHAGE LYSOGENIZING THE MARINE CYANOPHYTE PHORMIDIUM PERSICINUM1

A temperate cyanophage was found to lysogenize the marine cyanophyte Phormidium persicinum (Reinke) Com. (Provasoli strain). The lytic cycle was induced by the addition of mitomycin C or by brief illumination with ultraviolet light. The lytic process observed under the electron microscope showed that phage particles appeared in a nucleoplasm region 15 to 24 h after the addition of mitomycin C. The induction of the lytic process occurred simultaneously in almost all cells of every trichome. Matured phage particles were released to the medium 30 to 50 h after the addition of mitomycin C. Phage particles isolated from algal lysates had a polyhedral head (about 40 nm in diameter) with a long (about 300 nm) and noncontractile tail. The most abundant protein, presumably a structural protein, had an apparent molecular mass of about 38 kDa. The genome size estimated from restriction analysis was about 50 kbp. Phage DNA was digested with several restriction endonucleases including Sau3AI and DpnI. However, MboI failed to digest the phage DNA, suggesting that the phage DNA is highly methylated. Southern blot analysis suggested that some part of the phage was in the lytic cycle in algal cells growing under normal conditions. A possible role of temperate cyanophages in the regulation of cyanophyte populations in the marine environment is discussed.     

Biochemical and physiological characterisation of Erwinia species causing tip-over disease of banana

Tip-over disease has become a serious threat to banana plantations in the past decade. The disease is reported to be caused by Erwinia carotovorasubsp. carotovora and Erwinia chrysanthemi. We compared nine Erwinia strains of diseased banana plants from different agroclimatic zones of Karnataka and Andhra Pradesh, Southern India by conventional means. On the basis of morphological, cultural, physiological and biochemical characteristics and pathogenicity tests, the seven isolates I₁ to I₆ and I₈ showed similarities to Erwinia carotovorasubsp. carotovora. Isolate I₉ from Andhra Pradesh expressed characteristics similar to that of Erwinia chrysanthemi and was identified as Erwinia chrysanthemi. The isolate I₇ which showed wider variation, neither confirmed to the characteristics of Erwinia carotovorasubsp. carotovora nor with that of Erwinia chrysanthemi, and possessed characteristics in between the two species. Further we studied the host range of the bacterium causing tip-over disease of banana.     

Population dynamics ofAzospirillum brasilense and its bacteriophage in soil

Summary This study examined the ecology and interaction ofAzospirillum brasilense and its bacteriophage in soil. Four Chernozemic soils from Canada, a Latosol and three Podzolic soils from Brazil were assayed for phage. Only the Latosol containedA. brasilense phage. None of the soils contained phage for otherA. brasilense orA. lipoferum strains tested. Recovery of phage from soil depended on the growth of indigenous or added host cells. A phage isolated from the Latosol had a hexagonal head of 100 nm and a tail of 200 nm. This phage was morphologically distinct from previously described Azospirillum phage and its host range was limited toA. brasilense strains 29145 and 29711.Survival and recovery of phage added to phage-free soil was dependent on the phage, the initial phage population, the presence of host cells and nutrients, and the soil. Phage persisted in soils at undetectable levels for at least seven weeks, but were still able to interact with multiplying host cells and exhibit a 1000-fold increase in number. Phage required a host cell population of at least 100–1000 per g of soil in order to multiply. The phage burst detected under these conditions increased as the cell to phage ratio increased. Long term incubation studies showed that the activity of phage in soil closely followed the activity of host cells and thus both were manipulated by appropriate amendments to soil.     

Low pH and cold temperature combine to limit growth and pectate lyase production by the psychrotrophic bacterium Erwinia carotovora ssp. carotovora MFCL0

Growth and pectate lyase production by Erwinia carotovora ssp. carotovora MFCL0 were optimal at 28 °C and 14 °C, respectively. Although cold conditions (8 °C ) have retarded bacterial growth, low temperatures were not sufficient to prevent enzyme production but can be combined with a low pH (5.2) to protect vegetables against this phytopathogen.     

The Study of Erwinia carotovora Bacteriocins with the Aid of Nalidixic Acid–Resistant Bacterial Indicator Cells

A novel approach is proposed for the study of the macromolecular bacteriocins of Erwinia carotovora (MCTVs). The approach lies in the bacteriocinogeny of pectolytic erwinia being studied using a lawn of a bacterial mutant resistant to nalidixic acid, an inducer of MCTVs. The high efficiency of this approach was demonstrated by studying carotovoricins in 104 different E. carotovora strains, 88% of which bear MCTVs, distinguished by the morphology of zones of induced lysis on a lawn of susceptible cells, the lysis pattern, and some other characteristics. Preliminary studies using this approach showed that there is no correlation between the occurrence of MCTVs in particular E. carotovora strains and the habitat of the host plants from which these strains were isolated. There are grounds to believe that the approach proposed can also be used for investigating bacterial lysogeny.     

Expression of the Hypersensitive Response-assisting Protein in <Emphasis Type="Italic">Arabidopsis</Emphasis> Results in Harpin-dependent Hypersensitive Cell Death in Response to <Emphasis Type="Italic">Erwinia carotovora</Emphasis>

Active defense mechanisms of plants against pathogens often include a rapid plant cell death known as the hypersensitive cell death (HCD). Hypersensitive response-assisting protein (HRAP) isolated from sweet pepper intensifies the harpin_Pss-mediated HCD. Here we demonstrate that constitutive expression of the hrap gene in Arabidopsis results in an enhanced disease resistance towards soft rot pathogen, E. carotovora subsp. carotovora. This resistance was due to the induction of HCD since different HCD markers viz. Athsr3, Athsr4, ion leakage, H₂O₂ and protein kinase were induced. One of the elicitor harpin proteins, HrpN, from Erwinia carotovora subsp. carotovora was able to induce a stronger HCD in hrap-Arabidopsis than non-transgenic controls. To elucidate the role of HrpN, we used E. carotovora subsp. carotovora defective in HrpN production. The hrpN⁻ mutant did not induce disease resistance or HCD markers in hrap-Arabidopsis. These results imply that the disease resistance of hrap-Arabidopsis against a virulent pathogen is harpin dependent.     

Behavior of bacteriophage P1 inErwinia carotovora subsp.carotovora

Bacteriophage P1KMclr100 was tranferred toErwinia carotovora subsp.carotovora. P1 was stably maintained as detected by hybridization and transfer of kanamycin resistance. Lysogens ofE. carotovora failed to produce any viable P1 phage. Although total DNA from P1 lysogens ofE. carotovora hybridized to³²P-labeled P1 probe, we were not able to detect P1 DNA as an extrachromosomal element. Attempts to use bacteriophage P1 as a vector for transposon Tn5 insertion mutagenesis inE. carotovora were not successful. Our results indicate that lytic replication of P1 DNA does not occur in P1 lysogens ofE. carotovora and that P1 DNA is probably integrated into the bacterial chromosome.Journal paper 10085 from the Purdue Agricultural Experiment Research Station.     

一株感染铜绿假单胞菌的双链RNA噬菌体PaP6的分离和鉴定

【目的】鉴定一株新分离的铜绿假单胞菌噬菌体PaP6的生物学特性。【方法】利用铜绿假单胞菌临床分离株PA038为宿主,从西南医院污水中分离得到一株裂解性噬菌体PaP6,观察其噬斑特点;氯化铯密度梯度离心纯化噬菌体颗粒后,用透射电子显微镜观察噬菌体形态;提取PaP6基因组,通过DNA酶和RNA酶酶切,做基因组酶切图谱分析;按照感染复数(MOI)分别为10、1、0.1、0.01、0.001和0.000 1加入噬菌体和宿主菌,裂解细菌后,测定噬菌体滴度;以MOI=10的比例加入噬菌体和宿主菌,绘制一步生长曲线;用112株铜绿假单胞菌临床分离株检测PaP6宿主谱。【结果】PaP6的噬斑直径约2 mm-4 mm,圆形透明,边缘清晰;PaP6噬菌体呈多面体立体对称的头部,直径约45 nm;酶切图谱表明PaP6基因组对DNase不敏感,对RNase敏感,未酶切基因组具有3节段双链RNA(dsRNA),长度分别约为9.0、4.5、3.5 kb,共约17 kb;当MOI为0.1时PaP6感染其宿主菌产生的子代噬菌体滴度最高,达到3.4×109 PFU/m L;用一步生长曲线描绘了其生长特性;PaP6可以感染40.1%的临床分离株,是一株比较广谱的噬菌体。【结论】首次报道了一株铜绿假单胞菌的ds RNA分节段噬菌体,分类学上属于囊病毒科,该噬菌体具有较广的宿主谱,在噬菌体治疗领域具有应用前景。     

Cloning of the cysB gene of Erwinia carotovora subsp. carotovora, and the identification of its product

Summary The suicide vector pJB4JI was used to generate a range of Tn5-induced mutants of Erwinia carotovora subsp. carotovora (Ecc). One mutant, HC500, was a cysteine auxotroph which had a non-pectolytic, non-cellulolytic, non-proteolytic phenotype when grown under sulphate-limitation. The cysteine lesion of HC500 was shown to be analogous to the cysB mutation of Escherichia coli. The Ecc-cysB ⁺ gene product was identified as a protein of M_r 36000.     

Isolation and Preliminary Characterization of Cryptic Plasmids from Erwinia carotovora

Of the fifty-two Erwinia carotovorastrains studied, sixteen were found to contain extrachromosomal DNA (plasmids) from 2.5 to 129 kbp in size. Some E. carotovorastrains bore two to five different plasmids. Experiments showed that the cryptic plasmids of erwinia are not responsible for their resistance to antibiotics and are not involved in the synthesis of macromolecular colicin-like carotovoricins. At the same time, one of the E. carotovorastrains, 13A, augmented the production of carotovoricin after curing from one of its plasmids, the 47.7-kbp pCA 6-2. Three E. carotovorasubsp.carotovorastrains and one E. carotovorasubsp.atrosepticastrain contained large 129-kbp plasmids, which may play a role in the ecology of phytopathogenic pectinolytic erwinia.