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.     

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.     

Expression of klebsiella his and nif genes in serratia marcescens,Erwinia herbicola and Proteus mirabilis

Plasmid pRD1, an R plasmid of the P incompatibility group which carries his and nif genes from Klebsiella pneumoniae in addition to drug resistance markers derived from RP4, was transferred to His^- mutants of Serratia marcescens, Erwinia herbicola and Proteus mirabilis. His⁺ transconjugants were obtained at low but different frequencies according to recipient genus. Transconjugants all acquired the drug resistance, and were Nif⁺ in S. marcescens and E. herbicola, having acetylene-reducing activities of the same order of magnitude as the parent K. pneumoniae and fixing ¹⁵N₂. No evidence for nif expression in P. mirabilis transconjugants was obtained though the nif genes were present.     

Characterization of a genomic clone of Erwinia carotovora subsp. carotovora TG3 encoding a pectolytic enzyme of apparent molecular weight 78 kDa

Summary A clone containing the gene encoding a pectolytic enzyme of Erwinia carotovora subsp. carotovora was selected as the one that showed maceration on a solid medium containing sodium polypectate. The gene was located on a 3.2-kb DNA fragment flanked by a BglII site and a Hin-dIII site. Via mini-Mudlac mutagenesis, a possible promoter site was located within the gene between the BglII site and the EcoRI site. The mRNA transcribed from the promoter was directed from the BglII site toward the EcoRI site, determined from the orientation of the inserted mini-Mudlac. The probable gene product was identified as a 78 kDa protein. The enzyme activity of the Escherichia coli clone was detected mainly in the periplasmic space. Potato tuber slices were not macerated by the E. coli clone and synthesis of the enzyme in E. coli was not regulated by the enzyme substrate, sodium polypectate.     

Structure and regulation of the Erwinia carotovora subspecies carotovora SCC3193 cellulase gene celV1 and the role of cellulase in phytopathogenicity

The ceIV1 gene encoding a secreted cellulase (CelV1) of Erwinia carotovora subsp. carotovora SCC3193 was cloned and its nucleotide sequence determined. The gene contains an open reading frame of 1511 by and codes for an exported protein of 504 amino acids. The predicted amino acid sequence of Ce1V1 was highly similar to that of CeIV of another E. c. subsp. carotovora strain SCRI193 but completely different from the previously characterized cellulase, CelS, of the strain SCC3193. Gene fusions to the lacZ reporter were employed to characterize the regulation of celV1 and celS. Both genes are coordinately induced in a growth phase-dependent manner and are catabolite repressed. Expression of celV1 but not celS was stimulated by plant extracts. The celS gene was expressed at a much lower level than celV1 under all conditions tested. Inactivation of the celV1 gene in E. c. subsp. carotovora strain SCC3193 by marker exchange showed that celV1 encodes the major cellulase of strain SCC3193, as the resulting mutant strain SCC6001 was devoid of cellulase activity. Ce1Vl mutants exhibited reduced virulence suggesting that CelV1, although not absolutely required for pathogenicity, enhances the ability of strain SCC3193 to macerate plant tissue. Inactivation of the celS gene in the celV1 mutant did not lead to any further decrease in virulence.     

Isolation and characterisation of transposon-induced mutants of Erwinia carotovora subsp. atroseptica exhibiting reduced virulence

Summary The blackleg pathogen Erwinia carotovora subsp. atroseptica (Eca) causes an economically important disease of potatoes. We selected a genetically amenable Eca strain for the genetic analysis of virulence. Tn5 mutagenesis was used to generate nine mutants which exhibited reduced virulence (Rvi^-) of strain SCRI1043. Following physiological characterisation, mutants were divided into three classes: (1) auxotrophs; (2) extracellular enzyme mutants; and (3) a growth rate mutant. The isolation of these Rvi^- mutants has allowed us to consider some factors that affect Eca virulence.     

Conjugal transfer and expression of streptococcal transposons in Clostridium acetobutylicum

The transposon-containing streptococcal plasmids pAM211, pCF10, and pINY1275 have been transferred at high frequency (10^-2–10^-3 per recipient, selecting for tetracycline resistance) to the Gram-positive anaerobe Clostridium acetobutylicum. Selection in the presence of two antibiotics (tetracycline and erythromycin) with the plasmids pAM 180 and pINY1275 yielded only low numbers of transconjugants (10^-8 per recipient). Matings were done by combining liquid and filter mating procedures under anaerobic conditions. No plasmid DNA could be detected in the transconjugants selected on a minimal medium in the presence of tetracycline. DNA-DNA hybridization experiments with restricted chromosomal DNA using biotinylated pAM120::Tn916 as probe revealed the presence of homologous sequences in the transconjugants but not in Clostridium acetobutylicum wild type. The transconjugants were used as donors in mating experiments with tetracycline-sensitive Bacillus subtilis and Streptococcus lactis subspec. diacetylactis. In both cases tetracycline-resistant strains were found. Transfer frequencies in these experiments were less than 10^-7 per recipient.     

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.     

Molecular cloning and transposition of a kanamycin resistance determinant from Campylobacter jejuni between replicons in Escherichia coli

This paper reports a restriction map of a fragment of DNA encoding kanamycin resistance cloned from plasmid DNA of Campylobacter jejuni ABA94 in the recombinant plasmid pRS9421-1. In transposition experiments, kanamycin-resistant R751::km9421 transconjugants appeared at frequencies of 10^-7 per donor cell. These transconjugants harboured a plasmid 4 kb larger than the parental 49 kb plasmid R751. Restriction enzyme analysis and Southern blot hybridization of these transconjugants showed that the kanamycin resistant determinant had transposed from recombinant plasmid pRS9421-1 to plasmid R751.The authors are with the Department of Genetics and Cellular Biology, Faculty of Science, University of Malaya, 59100 Kuala Lumpur, Malaysia     

Production of soft rot resistant calla lily by expressing a ferredoxin-like protein gene (<Emphasis Type="Italic">pflp</Emphasis>) in transgenic plants

An efficient protocol for the Agrobacterium tumefaciens-mediated transformation of calla lily (Zantedeschia elliottiana (W. Wats.) Engl. cultivar ‘Florex Gold’) is described. Shoot basal discs were co-cultivated with A. tumefaciens C58C1 carrying a plasmid containing neomycin phosphotransferase (nptII) and plant ferredoxin-like protein (pflp) genes. After Agrobacterium co-cultivation, the shoot basal discs were exposed to 100 mg l⁻¹ kanamycin for selection. Twenty-eight out of 260 discs (10.8%) were found to have survived and produced shoot clusters. Twenty-six of these were confirmed to contain the pflp transgene by PCR, ending up in 10% transformation efficiency. The disease resistance investigation revealed that 18 transgenic plants exhibited resistance to soft rot disease caused by Erwinia carotovora subsp. carotovora. The presence of pflp gene was demonstrated by PCR, and its accumulation and activity was confirmed by Western blot and disease resistance assay. This was the first report to show the successful transformation and resistance to a bacterial pathogen in Zantedeschia. The protocol is useful for the quality improvement of calla lily through genetic transformation.     

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.     

Erwinia soft rot resistance of potato cultivars expressing antimicrobial peptide tachyplesin I

Tachyplesin I is a 2.3 kDa antimicrobial peptide isolated from Southeast Asian horseshoe crabs. Bacterial suspensions containing 1×10⁶ colony-forming units/ml of six isolates of pectolytic Erwinia spp., the causal pathogens of potato soft rot and blackleg, were killed in vitro by 1.4 to 11.1 g/ml of tachyplesin I. In an attempt to enhance resistance to Erwinia spp., each of the potato cultivars Bintje, Karnico and Kondor were transformed with two gene constructs encoding different precursor tachyplesin I proteins under the control of a cauliflower mosaic virus 35S promotor. Northern and western blot analysis showed that the tachyplesin I gene was expressed in transgenic plants. Small tubers of 17 transgenic clones were screened twice for soft rot resistance to Erwinia carotovora ssp. atroseptica. Under aerobic or anaerobic conditions, transgenic clones showed slightly less rot than control tubers.Abbreviations AP acidic carboxyl terminal polypeptide - Eca Erwinia carotovora ssp. atroseptica - Ecc E. carotovora ssp. carotovora - Ech E. chrysanthemi - IF intercellular fluid - SP signal peptide - TPNI (tpnI) tachyplesin I     

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.     

Transfer of an insecticidal protein gene ofBacillus thuringiensis into plant-colonizingAzospirillum

A fusion plasmid, pRKC, was constructed, using pACYC184, RSF1010 and a kanamycin-resistance cartridge from pUC4K, to convey thecryIA(a) gene intoAzospirillum spp. With the pRKC plasmid, the number of putative transconjugants obtained inA. lipoferum was about 300-fold higher than inA. brasilense. Conjugation frequency and plasmid stability inA. lipoferum were less for pBTF8, which carries thecryIA(a) gene in the correct orientation for a constitutive promoter, than for pBTF9, which carries the gene in the opposite orientation. Expression of thecryIA(a) gene was not apparent in SDS-PAGE analysis ofA. lipoferum transconjugants harbouring pBTF8. However,Escherichia coli transformants with the pBTF8 rescued fromA. lipoferum transconjugants produced an approximately 135 kDa Cry protein, indicating that thecry gene is intact in the transconjugants.V. Udayasuriyan was and A. Nakamura, H. Masaki and T. Uozumi are with the Department of Biotechnology, Faculty of Agriculture, The University of Tokyo, Yayoi 1-1-1, Bunkyo-Ku, Tokyo 113, Japan; V. Udayasuriyan is now with the Department of Plant Molecular Biology and Biotechnology, Tamil Nadu Agricultral University, Coimbatore-641 003, India.     

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.     

Screening Antimicrobial Activities of Basic Protein Fractions from Dry and Germinated Wheat Seeds

Two small cationic peptide fractions (5 kDa) were isolated from dry and germinated seeds of wheat, named WAP and GWAP, respectively. The antifungal and antibacterial activities of the peptides were analyzed using disk diffusion and turbidity measurement assays. The peptides in vitro exhibited effective antifungal activity against four plant pathogenic fungi at minimum concentration of 15 g(protein) cm^–3. Their antimicrobial activity was negatively affected by the presence of 5 mM CaCl₂. The peptides were less effective against Gram-negative bacterium Erwinia carotovora subsp. carotovora, but they demonstrated inhibitory activity against Gram-positive bacterium Clavibacter michiganensis subsp. sepedonicus. The antimicrobial activity of GWAP was more effective than WAP.     

Continuous-flow enrichment of a strain ofErwinia carotovora having specificity for highly methylated pectin

A pectinolytic bacterium was isolated from a mixed microbial population by means of a chemostat enrichment procedure. The bacterium, which was identified asErwinia carotovora, grew only on highly methylated pectin and produced a pectin lysase which released unsaturated monomer and dimer from 71% esterified citrus pectin. The pectin lyase was inducible only by pectins having a high methyl content and in pectin-limited chemostats its synthesis passed through a maximum at a dilution rate close to 0.04h^-1.     

母乳源乳双歧杆菌Probio-M8的遗传特征及基因组差异比较分析

【目的】母乳源乳双歧杆菌（Bifidobacterium animalis subsp. lactis） Probio-M8具有优良的益生特性,本文拟从全基因组水平解析Probio-M8的遗传特征,并与已有益生功效的乳双歧杆菌的基因组进行比较分析。【方法】本研究基于NCBI已公开的21株乳双歧杆菌和1株模式菌株DSM10140^T的基因组数据,构建了核心基因集与泛基因集,解析该群体的系统发育关系,比较分析Probio-M8的遗传特征及功能基因组。【结果】22株乳双歧杆菌的泛基因集包含1 618个基因,其中核心基因1 514个,占泛基因集的93.57%,表明乳双歧杆菌核心基因集高度保守。以1 514个核心基因构建系统发育树,发现22株乳双歧杆菌分为两个分支,AD011单独为一个分支,Probio-M8和其他菌株与模式菌株DSM10140^T聚在同一分支,且Probio-M8与V9、BB-12、Bi-07、HN019的遗传距离极为接近。进一步分析耐药基因和毒力基因,在Probio-M8与V9、BB-12、Bi-07、HN019基因组上均检测到DfrA...     

Expression ofKlebsiella pneumoniae nif genes inProteus mirabilis

Self-transmissible plasmids carryinghis andnif genes fromKlebsiella pneumoniae have been introduced into threehis mutants ofProteus mirabilis: strains 5006-1, WR19 and WR20. Expression ofhis by the transconjugants was unequivocal, if slightly temperature-sensitive, but none was Nif⁺ when tested for acetylene reduction in anaerobic glucose medium using inocula from rich or glucose-minimal aerobic agar cultures. Succinate or pyruvate in place of glucose, low glucose, lower temperature or elevated Na₂MoO₄ did not allownif expression and no nitrogenase MoFe-protein peptide was detected immunologically after exposure to conditions in which diazotrophic enterobacteria, normal or genetically constructed, derepressnif.One strain,P. mirabilis WR19, carrying thehis nif Km^r plasmid pMF250 was examined in detail. Thenif activator genenifA was introduced on the plasmid pCK1. Such derivatives remained Nif^- when tested, after aerobic growth on rich agar media, with normal or low glucose, with succinate or with elevated Mo. However, pre-conditioning by aerobic growth on glucose-minimal agar led to subsequent anaerobic expression ofnif in glucose medium from pMF250 in WR19 carrying pCK1. NH ₄ ⁺ or proline could serve as N-source in the glucose-minimal agar. Maximum activity was about 5% of that ofK. pneumoniae in our assay conditions. Material cross-reacting with anti-serum to the nitrogenase MoFe protein was formed. Nitrogenase activity was not switched off by NH ₄ ⁺ .P. mirabilis WR19 (pCK1) showed NH ₄ ⁺ -constitutive temperature-sensitive kanamycin resistance (anif-related phenotype of this plasmid) in aerobic glucose minimal medium. Expression ofnif inP. mirabilis WR19 (pCK1, pMF250) was NH ₄ ⁺ -repressible despite the constitutivenifA character of pCK1 and introduction of thentrA ⁺ plasmid pMM17 did not alter this phenotype. However, pCK1 did not give rise to NH ₄ ⁺ -constitutive diazotrophy in the wild-typeK. pneumoniae M5al. A construct of WR19 carrying pMF250 and constitutiventrC plasmid (pMD45) remained Nif^- even after pre-growth on glucose-minimal media.We conclude (a) thatP. mirabilis forms a gene product functionally equivalent to that ofntrA inK. pneumoniae, (b) that it forms no functional equivalent of thentrC product in our growth conditions. The need for pre-conditioning on aerobic glucose media remains perplexing.Non-common abbreviation NFDM Nitrogen-free-Davis-Mingioli medium     

Effective plasmid pX3 transduction in Lactobacillus delbrueckii by bacteriophage LL-H

High-frequency plasmid transductions in Lactobacillus delbrueckii subsp. lactis and subsp. bulgaricus strains mediated by pac-type bacteriophages were observed and further investigated. The frequency of plasmid transduction by phages LL-H and LL-S attained levels of from 0.10 to about 1 with plasmid pX3, but only about 2 × 10⁻² with plasmid pJK650. Infection of L. delbrueckii subsp. lactis strain LKT(pX3) or ATCC 15808(pX3) with phage LL-H resulted in intensive concatemerization of plasmid pX3, and most progeny phage particles contained concatemers of plasmid DNA instead of phage LL-H DNA. The synthesis of phage LL-H DNA was depressed. No evident homology or recombination was observed between phage LL-H DNA and plasmid pX3. The unusually high frequency of plasmid pX3 transduction by phage LL-H could be considered to result from specific interaction(s) between a particular phage and plasmid. These interactions may include pX3-mediated blockage of phage LL-H DNA replication and effective use of a particular pac-like site located about 1 kb from BglII in the smaller NdeI-BglII fragment of plasmid pX3. Phage LL-H together with plasmid vector pX3 could be used as effective plasmid transduction tools for genetic engineering of L. delbrueckii subsp. lactis and subsp. bulgaricus strains.