Restriction-modification systems determined by Pseudomonas plasmids

Four additional Pseudomonas R plasmids determining the PaeR7 restriction-modification system have been detected. All are transfer deficient and appear to belong to the same incompatibility group. The Pseudomonas fertility plasmid FP110 determines a different restriction-modification system and also inhibits the propagation of phage B39 by a separate mechanism. Pseudomonas R plasmid pMG73 has a third distinct restriction-modification specificity. PaeFP110 and PaeR73 are proposed as designations for these new plasmid-determined systems for restriction and modification.     

The restriction endonucleases in Bacillus amyloliquefaciens N strain. Substrate specificities.

Two species of restriction endonuclease were isolated by gel filtration and DEAE-cellulose chromatography from a cell-free extract of Bacillus amyloliquefaciens (B. subtilits) N strain; a lower molecular weight endonuclease (endonuclease R.BamNI) and a higher molecular-weight one (endonuclease R.BamNx). Both of them required only Mg2+ for their activities. Endonuclease R.BamNx introduced a larger number of site-specific scissions in Excherchia coli phage lambda DNA that endonuclease R.BamNI did. Endonuclease R.BamNx cleaved Bacillus phage phi 105C DNA at the specific sites which are classified into two groups: one type of sites is modified by B. amyloliquefaciens H strain in vivo while the other is not affected. It was also active on DNA'S OF E. coli phage T7, lambdadvl, Simian virus 40 (SV40) and colicinogenic factor ColEI and was inactive on DNAs of Bacillus phages phi 29 and M2. Endonuclease R.BamHI isolated from H strain by Wilson and Young. This endonuclease was active on DNAs of phage lambda, lambdadvl and SV40, adn was inactive on DNAs of phages phi 105C, phi 29, M2 and T7, and ColEI DNA.     

Restriction cleavage maps of the DNAs of Streptococcus pneumoniae bacteriophages containing protein covalently bound to their 5'' ends

Summary Several pneumococcal bacteriophages showing a morphology similar to that previously described for Cp-1 (Ronda et al. 1981) have been isolated and purified from throat samples taken from healthy children. Three of these phages (Cp-5, Cp-7 and Cp-9) have been studied in detail and compared to Cp-1. The four phages differed in several respects, e.g. size, structural polypeptides, restriction enzyme cleavage patterns, etc. The DNA of Cp-5, Cp-7 and Cp-9 showed protease-sensitive transfecting activity. This, together with the results obtained by electrophoretic analyses as well as by isotopic labelling of these DNAs with [-³²P] ATP and polynucleotide kinase indicated that all these new phages have a protein covalently linked to the 5 ends of their DNAs as in the case of Cp-1 (García et al. 1983). Restriction enzyme cleavage maps of Cp-1, Cp-5, Cp-7 and Cp-9 have been constructed.     

Isolation and characterization of lambda phages carrying the basic replicon of the resistance plasmid R1

Summary Specialized transducing lambda phages, oriR1, harboring DNA from the resistance plasmid R1drd-19 and its copy mutant pKN103 were isolated. From measurements of CCC-DNA content it is concluded that upon infection the phages can establish themselves as self-replicating plasmids in recA hosts lysogenic for lambda. It is thought that this bypassing of lambda immunity is due to the presence of the R1 origin of replication. The plasmids are sensitive to the incompatibility expressed by plasmid R1. This has been shown mainly by transduction of oriR1 into recipients containing R1 plasmids or plasmid pBR322 carrying the basic replicon. We were able to demonstrate that a copy mutant of plasmid R1 was insensitive to copA ⁺, but sensitive to the conserted action of Pst1 fragments F₁ and F₂. This mutant was previously assumed to be of the dominant type. Physical mapping of the oriR1 derivatives verified that they carry the basic replicon of plasmid R1. The plasmids are not stably maintained, but are lost in a frequency of 1%–2% per cell generation, which is consistent with their lack of the R1par region.     

Isolation and Molecular Characterization of pMG160, a Mobilizable Cryptic Plasmid from Rhodobacter blasticus

A 3.4-kb cryptic plasmid was obtained from a new isolate of Rhodobacter blasticus. This plasmid, designated pMG160, was mobilizable by the conjugative strain Escherichia coli S17.1 into Rhodobacter sphaeroides, Rhodobacter capsulatus, and Rhodopseudomonas palustris. It replicated in the latter strains but not in Rhodospirillum rubrum, Rhodocyclus gelatinosus, or Bradyrhizobium species. Plasmid pMG160 was stably maintained in R. sphaeroides for more than 100 generations in the absence of selection but showed segregational instability in R. palustris. Instability in R. palustris correlated with a decrease in plasmid copy number compared to the copy number in R. sphaeroides. The complete nucleotide sequence of plasmid pMG160 contained three open reading frames (ORFs). The deduced amino acid sequences encoded by ORF1 and ORF2 showed high degrees of homology to the MobS and MobL proteins that are involved in plasmid mobilization of certain plasmids. Based on homology with the Rep protein of several other plasmids, ORF3 encodes a putative rep gene initiator of plasmid replication. The functions of these sequences were demonstrated by deletion mapping, frameshift analysis, and analysis of point mutations. Two 6.1-kb pMG160-based E. coli-R. sphaeroides shuttle cloning vectors were constructed and designated pMG170 and pMG171. These two novel shuttle vectors were segregationally stable in R. sphaeroides growing under nonselective conditions.     

Identification of a New 1,4-beta-D-xylosidase <Emphasis Type="Italic">Pae</Emphasis>1263 from the Whole Genome Sequence of <Emphasis Type="Italic">Paenibacillus terrae</Emphasis> HPL-003

The gene of Pae1263 (2,196 bp, 732 aa) was found from the full-length sequence analysis of bacterium Paenibacillus terrae HPL-003 isolated from soil on Gara Mountain in Korea (CP003107, our previous study). Among the 20 open reading frames (ORFs) related with the xylose substrate, only the recombinant enzyme of ORF Pae1263 showed a 1,4-beta-D-xylosidase activity when all of the ORFs were transformed into E. coli. This gene is considered to be a new 1,4-beta-D-xylosidase because it has up to 93% similarity with other genes of ZP_10240221.1 from Lactococcus raffinolactis 4877 and ZP_11237858.1 from Paenibacillus peoriae in the GenBank blast search. The enzyme activity was confirmed by HPLC in which xylose was produced from xylobiose as a substrate by this recombinant enzyme. Mass production of the recombinant enzyme was done with the construction of the pET22(+)- Pae1263-6H expression vector system from E. coli. This new 1,4-beta-D-xylosidase was highly active at 50°C in a pH range between 6.0 and 8.0 and had thermo-stability for at least 24 h at 50°C and a K _m and V _max of 6.42 mg/mL and 75.76 U/mg on a xylobiose substrate, respectively.     

Dependence of the Genotypic Characteristics of <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis> on the Physical,Chemical, and Electrophysical Properties of Pyrites

This study focused on the effect of physical, chemical, and electrophysical properties of two pyrites, pyrite 1, which had electron-type (n-type) conductivity, and pyrite 2, with hole-type (p-type) conductivity, on the genotypic characteristics of Acidithiobacillus ferrooxidans strains TFV-1 and TFBk, which were isolated from different substrates. After the adaptation of the strains to the pyrites at a pulp density of 1%, pulsed-field electrophoresis revealed changes in the chromosomal DNA of strain TFV-1 adapted to pyrite 1, and strain TFBk adapted to either of the pyrite types. In pyrite-adapted strain TFBk, the plasmid composition was the same as after growth on a medium containing ferrous iron, whereas, in strain TFV-1, changes in plasmid sizes or both in plasmid sizes and plasmid number occurred. After an increase in the density of the pyrite 2 pulp from 1 to 10%, the plasmid number increased from three to four, and, after an increase in the density of the pyrite 1 pulp from 1 to 7%, the plasmid number increased from two to six.     

The Linear Plasmid Prophage Vp58.5 of Vibrio parahaemolyticus Is Closely Related to the Integrating Phage VHML and Constitutes a New Incompatibility Group of Telomere Phages

Vibrio parahaemolyticus O3:K6 pandemic strains recovered in Chile frequently possess a 42-kb plasmid which is the prophage of a myovirus. We studied the prototype phage VP58.5 and show that it does not integrate into the host cell chromosome but replicates as a linear plasmid (Vp58.5) with covalently closed ends (telomeres). The Vp58.5 replicon coexists with other plasmid prophages (N15, PY54, and ΦKO2) in the same cell and thus belongs to a new incompatibility group of telomere phages. We determined the complete nucleotide sequence (42,612 nucleotides) of the VP58.5 phage DNA and compared it with that of the plasmid prophage. The two molecules share the same nucleotide sequence but are 35% circularly permuted to each other. In contrast to the hairpin ends of the plasmid, VP58.5 phage DNA contains 5′-protruding ends. The VP58.5 sequence is 92% identical to the sequence of phage VHML, which was reported to integrate into the host chromosome. However, the gene order and termini of the phage DNAs are different. The VHML genome exhibits the same gene order as does the Vp58.5 plasmid. VHML phage DNA has been reported to contain terminal inverted repeats. This repetitive sequence is similar to the telomere resolution site (telRL) of VP58.5 which, after processing by the phage protelomerase, forms the hairpin ends of the Vp58.5 prophage. It is discussed why these closely related phages may be so different in terms of their genome ends and their lifestyle.Most temperate bacteriophages integrate into the host chromosome during lysogeny. However, there are some phages (telomere phages) whose prophages are linear plasmids with covalently closed ends. Members of this group of phages are the siphoviruses N15, PY54, and ΦKO2 isolated from Escherichia coli, Yersinia enterocolitica, and Klebsiella oxytoca, respectively, and the recently described myoviruses ΦHAP-1 of Halomonas aquamarina and VP882 of Vibrio parahaemolyticus (6, 20, 23, 26, 37). Despite their different origins (enterobacteria versus marine bacterium) and morphologies, all known telomere phages share similar genome organizations and some protein similarities. The linear DNA of each phage is a circular permutation of the respective linear plasmid prophage. For the generation of the terminal hairpins of the linear plasmid, the protelomerase (Tel) is essential (8). This enzyme has cleaving/joining activity; its target is a large palindromic DNA sequence called the telomere resolution site (telRL) located upstream of tel on the phage genome. After cleaving telRL by staggered cuts, the resulting self-complementary single-stranded DNA overhangs fold back and are rejoined by the protelomerase (9). Besides tel, all telomere phages possess the gene repA, encoding a multifunctional replication protein. repA of N15 and PY54 was shown to harbor the prophage replication origin and to function as a circular minimal replicon (35, 42). Compatibility studies demonstrated that the N15 and ΦKO2 plasmids belong to the same incompatibility group, whereas the PY54 plasmid is able to coexist with these two prophages in doubly lysogenic E. coli and Y. enterocolitica hosts (19).There are some reports on the presence of tel and repA in prophages (VP882, VHML, and Vp58.5) of marine Vibrio strains (28, 41). V. parahaemolyticus phage VP882 is a close relative of the Halomonas phage ΦHAP-1 (26). VHML was isolated from a toxin-producing Vibrio harveyi strain, pathogenic for some crustaceans and fish (30). Similarly to ΦHAP-1 and VP882, VHML has a myovirus-like morphology. The phage contains genes for products similar to Tel and RepA, suggesting that its prophage is a linear plasmid with terminal hairpins. However, it was surmised that VHML integrates into the Vibrio chromosome (28, 29). Phage VP58.5 was isolated from a V. parahaemolyticus strain belonging to the serovar O3:K6 pandemic clonal complex (41). During the last several years, this clone has been associated with many seafood-borne diarrhea outbreaks in Southeast Asia and South America, particularly Chile (5, 12, 13, 15). Up to 33% of the Chilean isolates harbored a 42-kb plasmid which was shown to be the prophage of a myovirus inducible by mitomycin C. VP58.5 is the prototype of these phages.In this work we demonstrate that VP58.5 is closely related to the V. harveyi phage VHML but that its prophage is a linear plasmid with covalently closed ends. The Vp58.5 prophage belongs to a new incompatibility group of telomere phages.     

A Study of 33 Bacteriophages of Rhizobium meliloti

A total of 33 Rhizobium meliloti bacteriophages were studied. Of those, 21 were isolated in northern France from field soil in which Medicago sativa L. was grown. The other 12 phages were obtained by UV light and mitomycin C induction from 46 R. meliloti strains. Rhizobiophages were characterized by their morphology, host range, serological properties, restriction endonuclease patterns, DNA-DNA homologies, and DNA molecular weights. Five morphotypes were observed showing tailed phages with icosahedral heads. The categories of morphotypes included the Myoviridae (11 phages), Siphoviridae (3 morphotypes and 20 phages), and Podoviridae (2 phages). Type NM1 phage (Siphoviridae) is highly unusual because of the presence of transverse bars on the phage tail. Soil phages had broad host ranges, whereas phages isolated from bacterial cultures showed more or less narrow host ranges. Restriction endonuclease patterns and DNA-DNA hybridization experiments showed that the five phage type genomes were unrelated. Molecular weights of phage type DNAs were estimated, and they corresponded to values expected for capsid sizes, except for phage NM8. Type M11S (Siphoviridae) did not correspond to any other described Rhizobium phages and represents a new species.     

Effect of Increasing the Copy Number of Bacteriophage Origins of Replication, in trans, on Incoming-Phage Proliferation

Bacteriophage resistance mechanisms which are derived from a bacteriophage genome are termed Per (phage-encoded resistance). When present in trans in Lactococcus lactis NCK203, Per50, the cloned origin of replication from phage 50, interferes with 50 replication. The per50 fragment was found to afford negligible protection to NCK203 against 50 infection when present in a low-copy-number plasmid, pTRK325. A high-copy-number Per50 construct (pTRK323) dramatically affected 50 infection, reducing the efficiency of plaquing (EOP) to 2.5 × 10^-4 and the plaque size to pinhead proportions. This clone also afforded significant protection against other related small isometric phages. Per31 was cloned from phage 31 and demonstrated to function as an origin of replication by enabling replication of per31-containing plasmids, in NCK203, on 31 infection. A low-copy-number Per31 plasmid (pTRK360) reduced the EOP of 31 on NCK203 to 0.3 and the plaque diameter from 1.5 to 0.5 mm. When this plasmid was cloned in high copy number, the EOP was further reduced to 7.2 × 10^-7 but the plaques were large and contained Per31-resistant phages. Characterization of these “new” phages revealed at least two different types that were similar to 31, except that DNA alterations were noted in the region containing the origin. This novel and powerful abortive phage resistance mechanism should prove useful when directed at specific, problematic phages.     

Cloning of mglB, the structural gene for the galactose-binding protein of Salmonella typhimurium and Escherichia coli

Summary From libraries of EcoRI fragments of Salmonella thyphimurium and Escherichia coli DNA in gt7, phages could be isolated that carry mglB, the structural gene of the galactose-binding protein as well as other mgl genes. Lysogenization of an E. coli mutant carrying a defective galactose-binding protein with gt7 mglB (Salmonella) restores full galactose transport and galactose chemotaxis. Both the E. coli mutant protein as well as the wild-type Salmonella galactose-binding protein are synthesized in this strain. The EcoR1 fragments of both organisms carrying the mgl genes were 6 Kb long. They were subcloned into the multicopy plasmid pACUC184. The hybrid plasmid containing the Salmonella mgl DNA gives rise to the synthesis of large amounts of galactose-binding protein in the periplasm of E. coli. The protein can be precipitated by antibodies against the E. coli binding protein and is identical to the fully processed protein isolated from Salmonella typhimurium LT2. In vitro protein synthesis (Zubay-system) with either gt7 mgl phages as well as the hybrid plasmid as DNA matrix produces the galactose-binding protein mainly in precursor form that is precipitable by specific antibodies.     

High-Frequency Plasmid Transduction by Lactobacillus gasseri Bacteriophage φadh

The temperate bacteriophage adh mediates plasmid DNA transduction in Lactobacillus gasseri ADH at frequencies in the range of 10^-8 to 10^-10 transductants per PFU. BglII-generated DNA fragments from phage adh were cloned into the BclI site of the transducible plasmid vector pGK12 (4.4 kb). Phage adh lysates induced from Lactobacillus lysogens harboring pGK12 or the recombinant plasmids were used to transduce strain ADH to chloramphenicol resistance. The transduction frequencies of recombinant plasmids were 10²- to 10⁵-fold higher than that of native pGK12. The increase in frequency generally correlated with the extent of DNA-DNA homology between plasmid and phage DNAs. The highest transduction frequency was obtained with plasmid pTRK170 (6.6 kb), a pGK12 derivative containing the 1.4- and 0.8-kb BglII DNA fragments of adh. DNA hybridization analysis of pTRK170-transducing phage particles revealed that pTRK170 had integrated into the adh genome, suggesting that recombination between homologous sequences present in phage and plasmid DNAs was responsible for the formation of high-frequency transducing phage particles. Plasmid DNA analysis of 13 transductants containing pTRK170 showed that each had acquired intact plasmids, indicating that in the process of transduction a further recombination step was involved in the resolution of plasmid DNA monomers from the recombinant pTRK170::adh molecule. In addition to strain ADH, pTRK170 could be transduced via adh to eight different L. gasseri strains, including the neotype strain, F. Gasser 63 AM (ATCC 33323).     

Comparative Genomics of the IncA/C Multidrug Resistance Plasmid Family

Multidrug resistance (MDR) plasmids belonging to the IncA/C plasmid family are widely distributed among Salmonella and other enterobacterial isolates from agricultural sources and have, at least once, also been identified in a drug-resistant Yersinia pestis isolate (IP275) from Madagascar. Here, we present the complete plasmid sequences of the IncA/C reference plasmid pRA1 (143,963 bp), isolated in 1971 from the fish pathogen Aeromonas hydrophila, and of the cryptic IncA/C plasmid pRAx (49,763 bp), isolated from Escherichia coli transconjugant D7-3, which was obtained through pRA1 transfer in 1980. Using comparative sequence analysis of pRA1 and pRAx with recent members of the IncA/C plasmid family, we show that both plasmids provide novel insights into the evolution of the IncA/C MDR plasmid family and the minimal machinery necessary for stable IncA/C plasmid maintenance. Our results indicate that recent members of the IncA/C plasmid family evolved from a common ancestor, similar in composition to pRA1, through stepwise integration of horizontally acquired resistance gene arrays into a conserved plasmid backbone. Phylogenetic comparisons predict type IV secretion-like conjugative transfer operons encoded on the shared plasmid backbones to be closely related to a group of integrating conjugative elements, which use conjugative transfer for horizontal propagation but stably integrate into the host chromosome during vegetative growth. A hipAB toxin-antitoxin gene cluster found on pRA1, which in Escherichia coli is involved in the formation of persister cell subpopulations, suggests persistence as an early broad-spectrum antimicrobial resistance mechanism in the evolution of IncA/C resistance plasmids.Antimicrobial compounds have been used extensively in agriculture since the 1960s not only to treat and prevent disease in plants, fruits, vegetables, and animals but also to promote growth in fish, poultry, and other livestock (42). The risk of transferring antimicrobial drug resistance to nonresistant bacteria and the propagation of multidrug-resistant (MDR) bacteria from agricultural to clinical and/or community-associated settings are being debated by research, regulatory, and health authorities (27, 28). In this context, the recent discovery of a group of self-transferable IncA/C antimicrobial resistance plasmids, which are widely distributed among agricultural nontyphoidal Salmonella enterica isolates from the United States (24, 45) has caused considerable concern in the public health community. Similar IncA/C plasmids were identified in an MDR isolate from Madagascar of Yersinia pestis, the causative agent of the plague (16), and MDR strains of Vibrio cholerae O139 from China (34), as well as in MDR isolates of the fish pathogen Photobacterium damselae subsp. piscicida from the United States and Japan (21). While the IncA/C group of MDR plasmids seems to be efficient in collecting antimicrobial resistance traits and mobilizing them across geographical and taxonomical borders, little is known about the evolutionary origin of these plasmids or the genetic basis for their spread.The IncA/C reference plasmid, pRA1, was isolated in 1971 from the fish pathogen Aeromonas liquefaciens, later renamed Aeromonas hydrophila, as a transferable antimicrobial resistance plasmid conferring resistance to sulfonamides and tetracyclines (2). The repA gene of pRA1, located at the origin of replication and responsible for encoding the replication initiation protein A, has been sequenced (25) and is used for PCR-based replicon typing of IncA/C plasmids (7). repA genes from all sequenced IncA/C plasmids to date share at least 98% nucleotide sequence identity.To better understand the evolutionary origin of IncA/C plasmids, pRA1 was isolated, sequenced, and compared to all IncA/C plasmid sequences currently available. In addition to pRA1, a pRA1-derived cryptic IncA/C plasmid, designated pRAx, was also sequenced and included in the analysis. pRAx was isolated from Escherichia coli D7-3, a strain that was obtained through the conjugative transfer of pRA1 from A. hydrophila in 1980 (30). While the laboratory history of the pRAx-carrying strain E. coli D7-3 since the conjugative plasmid acquisition is unknown, pRAx was included in this study as it tested positive for the repA reference gene from pRA1 (100% nucleotide sequence identity) but negative for 11 out of 12 additional IncA/C marker genes that were shown to be part of a conserved plasmid backbone shared by recently isolated IncA/C plasmids (45).     

Identification of restriction barriers in Pasteurella multocida

Several naturally occurring antibiotic resistance plasmids were isolated from Pasteurella multocida type D strains. One plasmid, pPM1, was used to study transfer of DNA among P. multocida strains, and could be transferred into Escherichia coli and some P. multocida isolates. However, pPM1 could only be transferred into the toxigenic P. multocida LFB3 at very low frequency. Plasmid recovered from the electrotransformants could be transferred to LFB3 at high frequency. These plasmid DNAs were resistant to PstI, and sensitive to DpnI digestion. Sensitivity to DpnI was common to all the P. multocida DNAs, but resistance to PstI was confined to LFB3. Plasmid pPM1 treated with PstI methylase was able to transform LFB3 at an increased frequency compared to unmethylated DNA, suggesting that LFB3 has a restriction system which cleaves at or near PstI sites.     

Sequence Analysis of the Cryptic Plasmid pMG101 from Rhodopseudomonas palustris and Construction of Stable Cloning Vectors

A 15-kb cryptic plasmid was obtained from a natural isolate of Rhodopseudomonas palustris. The plasmid, designated pMG101, was able to replicate in R. palustris and in closely related strains of Bradyrhizobium japonicum and phototrophic Bradyrhizobium species. However, it was unable to replicate in the purple nonsulfur bacterium Rhodobacter sphaeroides and in Rhizobium species. The replication region of pMG101 was localized to a 3.0-kb SalI-XhoI fragment, and this fragment was stably maintained in R. palustris for over 100 generations in the absence of selection. The complete nucleotide sequence of this fragment revealed two open reading frames (ORFs), ORF1 and ORF2. The deduced amino acid sequence of ORF1 is similar to sequences of Par proteins, which mediate plasmid stability from certain plasmids, while ORF2 was identified as a putative rep gene, coding for an initiator of plasmid replication, based on homology with the Rep proteins of several other plasmids. The function of these sequences was studied by deletion mapping and gene disruptions of ORF1 and ORF2. pMG101-based Escherichia coli-R. palustris shuttle cloning vectors pMG103 and pMG105 were constructed and were stably maintained in R. palustris growing under nonselective conditions. The ability of plasmid pMG101 to replicate in R. palustris and its close phylogenetic relatives should enable broad application of these vectors within this group of α-proteobacteria.     

Molecular Characterization of Three Small Isometric-Headed Bacteriophages Which Vary in Their Sensitivity to the Lactococcal Phage Resistance Plasmid pTR2030

Lactococcus lactis LMA12-4 is a pTR2030 transconjugant that has been used as an industrial starter culture because of its resistance to phages predominant in cheese plants. Plasmid pTR2030 interferes with susceptible phages in this host strain via two mechanisms, restriction and modification (R/M) and abortive infection (Hsp). After prolonged use of LMA12-4 transconjugants in the industry, two different bacteriophages, designated nck202.48 (48) and nck202.50 (50), were isolated which could produce plaques on LMA12-4 containing pTR2030. In this study, these two phages were characterized and compared with a third phage, nck202.31 (31), which is susceptible to both the R/M and Hsp activities encoded by pTR2030. Phage 48 was not susceptible to inhibition by Hsp, whereas 50 was unaffected by either the R/M or Hsp mechanisms. All three were small isometric-headed phages, but small differences were noted between the phages in the structural details of the tail base plate, susceptibility to chloroform treatment, and requirements for calcium infectivity. The phage genomes were all between 29.9 and 31.9 kb in length. Phages 31 and 48 harbored cohesive ends, whereas the phage 50 genome was circularly permuted, terminally redundant, and carried a putative packaging initiation site. DNA-DNA hybridization experiments conducted between the phages revealed a common region in 48 and 50 that may correlate with the resistance of the two phages to the Hsp-abortive infection induced by pTR2030. Phage 50 also harbored DNA sequences that shared homology to pTR2030 in the region where R/M activities have been localized on the plasmid. Molecular characterization of the three phages localized regions within the genomes of the pTR2030-resistant phages that may be responsible for circumventing plasmid-encoded Hsp and R/M defense mechanisms in lactococci.     

Comparative Genome Analysis Provides Insights into the Evolution and Adaptation of Pseudomonas syringae pv. aesculi on Aesculus hippocastanum

A recently emerging bleeding canker disease, caused by Pseudomonas syringae pathovar aesculi (Pae), is threatening European horse chestnut in northwest Europe. Very little is known about the origin and biology of this new disease. We used the nucleotide sequences of seven commonly used marker genes to investigate the phylogeny of three strains isolated recently from bleeding stem cankers on European horse chestnut in Britain (E-Pae). On the basis of these sequences alone, the E-Pae strains were identical to the Pae type-strain (I-Pae), isolated from leaf spots on Indian horse chestnut in India in 1969. The phylogenetic analyses also showed that Pae belongs to a distinct clade of P. syringae pathovars adapted to woody hosts. We generated genome-wide Illumina sequence data from the three E-Pae strains and one strain of I-Pae. Comparative genomic analyses revealed pathovar-specific genomic regions in Pae potentially implicated in virulence on a tree host, including genes for the catabolism of plant-derived aromatic compounds and enterobactin synthesis. Several gene clusters displayed intra-pathovar variation, including those encoding type IV secretion, a novel fatty acid biosynthesis pathway and a sucrose uptake pathway. Rates of single nucleotide polymorphisms in the four Pae genomes indicate that the three E-Pae strains diverged from each other much more recently than they diverged from I-Pae. The very low genetic diversity among the three geographically distinct E-Pae strains suggests that they originate from a single, recent introduction into Britain, thus highlighting the serious environmental risks posed by the spread of an exotic plant pathogenic bacterium to a new geographic location. The genomic regions in Pae that are absent from other P. syringae pathovars that infect herbaceous hosts may represent candidate genetic adaptations to infection of the woody parts of the tree.     

Mercury and Organomercurial Resistances Determined by Plasmids in Pseudomonas

Mercury and organomercurial resistance determined by genes on ten Pseudomonas aeruginosa plasmids and one Pseudomonas putida plasmid have been studied with regard to the range of substrates and the range of inducers. The plasmidless strains were sensitive to growth inhibition by Hg(2+) and did not volatilize Hg(0) from Hg(2+). A strain with plasmid RP1 (which does not confer resistance to Hg(2+)) similarly did not volatilize mercury. All 10 plasmids determine mercury resistance by way of an inducible enzyme system. Hg(2+) was reduced to Hg(0), which is insoluble in water and rapidly volatilizes from the growth medium. Plasmids pMG1, pMG2, R26, R933, R93-1, and pVS1 in P. aeruginosa and MER in P. putida conferred resistance to and the ability to volatilize mercury from Hg(2+), but strains with these plasmids were sensitive to and could not volatilize mercury from the organomercurials methylmercury, ethylmercury, phenylmercury, and thimerosal. These plasmids, in addition, conferred resistance to the organomercurials merbromin, p-hydroxymercuribenzoate, and fluorescein mercuric acetate. The other plasmids, FP2, R38, R3108, and pVS2, determined resistance to and decomposition of a range of organomercurials, including methylmercury, ethylmercury, phenylmercury, and thimerosal. These plasmids also conferred resistance to the organomercurials merbromin, p-hydroxymercuribenzoate, and fluorescein mercuric acetate by a mechanism not involving degradation. In all cases, organomercurial decomposition and mercury volatilization were induced by exposure to Hg(2+) or organomercurials. The plasmids differed in the relative efficacy of inducers. Hg(2+) resistance with strains that are organomercurial sensitive appeared to be induced preferentially by Hg(2+) and only poorly by organomercurials to which the cells are sensitive. However, the organomercurials p-hydroxymercuribenzoate, merbromin, and fluorescein mercuric acetate were strong gratuitous inducers but not substrates for the Hg(2+) volatilization system. With strains resistant to phenylmercury and thimerosal, these organomercurials were both inducers and substrates.