Identification of the replication region in pBCNF5603, a bacteriocin-encoding plasmid, in the enterotoxigenic Clostridium perfringens strain F5603.

BACKGROUND: Most recent studies of Clostridium perfringens plasmids have focused on toxin-encoding or antibiotic resistance plasmids. To cause intestinal disease, a toxigenic strain must grow in the intestines to levels allowing for sufficient toxin production and this in vivo growth often involves overcoming the normal intestinal microbial population. For this purpose, bacteriocin production might be important. RESULTS: In this study, as the first step in the genetic analysis of a co-existing plasmid with an enterotoxin gene (cpe)-encoding plasmid, the bacteriocin gene-encoding plasmid, pBCNF5603, was completely sequenced. This plasmid has some homology with two previously sequenced C. perfringens plasmids, namely, pCP13 carrying a cpb2 gene and pIP404 carrying a bcn gene. Using recombinant plasmids, the rep gene homologous to the PCP63 gene on pCP13 appeared to be functional. Comparative genomics indicated that the identified rep gene homologs were found on two additional toxin plasmids, pCP-OS1 and pCP-TS1. While functional analysis using recombinant plasmids indicated that pBCNF5603 and pCP13 are likely to be incompatible, the plasmid replication and partitioning region of pBCNF5603 alone was insufficient for stable maintenance of this plasmid. CONCLUSIONS: These findings suggest that pBCNF5603 evolved from recombination events between C. perfringens plasmids and inter-species mobile genetic element(s). In addition, the bcn-encoding plasmid, pBCNF5603, is likely to be included in the Inc family, which includes pCP13 and two variant iota-encoding plasmids. Furthermore, the bcn gene on pBCNF5603 could contribute to gastrointestinal disease induced by enterotoxigenic C. perfringens.

Accumulation of single-stranded DNA in Escherichia coli carrying the colicin plasmid pColE3-CA38.

We sequenced the complete 7118 bp circular plasmid pColE3-CA38 (pColE3) from Escherichia coli, located the previously identified colicin components together with two new ORFs that have homology to mobilization and transfer proteins, and found that pColE3 is highly similar to a plasmid present in enterohemorrhagic E. coli O111. We also found that unusual aspects of the plasmid include the inability to be completely digested with restriction endonucleases and asymmetric Phred DNA sequencing quality scores, with significantly lower scores in the forward direction relative to the colicin and immunity proteins consistent with plus (+) strand DNA. Comparing the A260 with picogreen double-stranded DNA (dsDNA) fluorescence and oligreen single-stranded DNA (ssDNA) fluorescence as well as metachromatic staining by acridine orange, we found that the undigested pColE3 DNA stains preferentially as ssDNA and that it coexists with dsDNA. We also identified ssDNA in pColE5 and pColE9 but not in pColE1. Colicin plasmids producing ssDNA may represent a new subclass of rolling-circle replication plasmids and add to the known similarities between colicins and filamentous phage.

Gut inflammation can boost horizontal gene transfer between pathogenic and commensal Enterobacteriaceae.

The mammalian gut harbors a dense microbial community interacting in multiple ways, including horizontal gene transfer (HGT). Pangenome analyses established particularly high levels of genetic flux between Gram-negative Enterobacteriaceae. However, the mechanisms fostering intraenterobacterial HGT are incompletely understood. Using a mouse colitis model, we found that Salmonella-inflicted enteropathy elicits parallel blooms of the pathogen and of resident commensal Escherichia coli. These blooms boosted conjugative HGT of the colicin-plasmid p2 from Salmonella enterica serovar Typhimurium to E. coli. Transconjugation efficiencies of ~100% in vivo were attributable to high intrinsic p2-transfer rates. Plasmid-encoded fitness benefits contributed little. Under normal conditions, HGT was blocked by the commensal microbiota inhibiting contact-dependent conjugation between Enterobacteriaceae. Our data show that pathogen-driven inflammatory responses in the gut can generate transient enterobacterial blooms in which conjugative transfer occurs at unprecedented rates. These blooms may favor reassortment of plasmid-encoded genes between pathogens and commensals fostering the spread of fitness-, virulence-, and antibiotic-resistance determinants.

Characteristics of Plasmids Coharboring 16S rRNA Methylases, CTX-M, and Virulence Factors in Escherichia coli and Klebsiella pneumoniae Isolates from Chickens in China.

The objective of this study was to characterize plasmids coharboring 16S rRNA methylases, blaCTX-M and virulence-associated genes in Escherichia coli and Klebsiella pneumoniae isolates from chickens in China. A total of 32 positive transconjugants exhibited coresistance to amikacin and cefotaxime in E. coli (24/281) and K. pneumoniae (8/93), and were identified by conjugation experiments and S1-pulsed-field gel electrophoresis. Polymerase chain reaction amplification assay detecting resistance genes showed that rmtB or armA gene accompanied with different blaCTX-M genes coexisted on 32 transferred plasmids. The blaCTX-M-98b gene was identified in chicken-derived E. coli and K. pneumoniae for the first time. The association between resistance genes and virulence genes was observed in the transferred plasmids; 68.8% (22/32) transferred resistance plasmids coharboring various virulence genes including traT, iutA, fyuA, msbB, and vagC genes with diverse proportions. Genetic stability tests revealed that 93.8% (30/32) transferred plasmids continued to exist in the host strain after continuous passage of 30 times in 15 days. Furthermore, 87.5% (28/32) conjugants showed no significant differences in growth rates compared with E. coli J53. Results of the growth competition assay showed that conjugants have low fitness cost, which indicated that there were no obvious negative effects on the host's growth. The combination of blaCTX-M-98b-rmtB-traT on 85-kb transferred IncF plasmids in E. coli, and blaCTX-M-14-rmtB-traT on 95-kb transferred IncF plasmids in K. pneumoniae were first identified in this study. These features of plasmids may contribute to the successful spread of resistance and virulence among pathogens of different sources and geographical origins.

Cloning of genes encoding colicin E2 in Lactococcus lactis subspecies lactis and evaluation of the colicin-producing transformants as inhibitors of Escherichia coli O157:H7 during milk fermentation.

Colicin E2 (ColE2) is a proteinaceous bacterial toxin produced by some strains of Escherichia coli and other members of the Enterobacteriaceae that exhibits inhibitory activity against some strains of E. coli O157:H7. A 2.0-kb DNA fragment, containing the ColE2 structural gene ceaB and immunity gene ceiB from E. coli NCTC 50133 (pColE2-P9), was cloned into the lactococcal plasmid vector pNZ2103. The lysis gene, celB, was not cloned. The plasmid, pLR-E2, encoding the cloned genes was transformed into E. coli DH5α and Lactococcus lactis ssp. lactis LM0230 and PN-1 using electroporation. The bacteriocin ColE2 was expressed in transformants of both E. coli and L. lactis ssp. lactis. Secretion of ColE2 into media was verified by spot-on-lawn assays and measurement of ColE2 activity in the growth medium of transformants. The level of ColE2 produced by transformants containing pLR-E2 was similar to that produced by the parental strain, E. coli NCTC 50133 (pColE2-P9). Evaluation of a ColE2-producing transformant of L. lactis ssp. lactis as a starter culture revealed that, although ColE2 was produced by transformants and could be detected in milk during fermentation, the inhibitory activity of ColE2 against E. coli O157:H7 was significantly decreased in milk compared with buffered growth medium.

The large plasmidome of Lactococcus lactis subsp. lactis bv. diacetylactis S50 confers its biotechnological properties.

Plasmids are autonomous episomally replicating genetic elements, which carry backbone genes important for the replication and maintenance within their host, and accessory genes that might confer an advantage to their host under specific selective pressure in its ecological niche. The genome of dairy isolate L. lactis subsp. lactis bv. diacetylactis S50 was sequenced using the PacBio SMRT Cell Seq-RSII platform and revealed to possess one of the largest plasmidomes among L. lactis strains studied so far, harboring six plasmids: pS6 (5553 bp), pS7a (7308 bp), pS7b (7266 bp), pS19 (19,027 bp), pS74 (74,256 bp) and pS127 (127,002 bp) in total representing 8.9% of genome size (240,412 bp). Based on predicted plasmid replication proteins and origins it appears that all six plasmids replicate via the theta-type mechanism. The two the largest plasmids (pS74 and pS127), carry a number of genes known to be important for growth and survival in the dairy environment. These genes encode technological functions such as bacteriocin production, protein degradation, magnesium and cobalt/nickel transporters, selenium binding, exopolysaccharides (EPS) production, bacteriophage and stress resistance. Beside genes for replication, the small plasmids (pS6, pS7a, pS7a, and pS19) also carry genes important for mobilization and host survival such as type I restriction-modification (R-M) system, metal transporters, enzymes and transcriptional regulators. All plasmids in S50 strain are mobilizable, containing an oriT sequences, while pS127 is self-conjugative and allows for mobilization of the other plasmids. Small plasmids are prone to structural and segregational instability, while pS127 appeared to be segregationally stable thanks to the possession of two partition systems. The main characteristic of plasmid pS74 is EPS production, while plasmid pS127 is characterized by proteinase and multiple bacteriocins, tra locus, phage abortive systems and metal transporters. In addition to LcnA and LcnB, plasmid pS127 encodes several bacteriocin-pheromone molecules and a new bacteriocin named LcnS50, with narrow spectrum of action limited to lactococci, that has been successfully cloned and heterologously expressed.

Insights into the functionality of the putative residues involved in enterocin AS-48 maturation.

AS-48 is a 70-residue, α-helical, cationic bacteriocin produced by Enterococcus faecalis and is very singular in its circular structure and its broad antibacterial spectrum. The AS-48 preprotein consists of an N-terminal signal peptide (SP) (35 residues) followed by a proprotein moiety that undergoes posttranslational modifications to yield the mature and active circular protein. For the study of the specificity of the region of AS-48 that is responsible for maturation, three single mutants have been generated by site-directed mutagenesis in the as-48A structural gene. The substitutions were made just in the residues that are thought to constitute a recognition site for the SP cleavage enzyme (His-1, Met1) and in those involved in circularization (Met1, Trp70). Each derivative was expressed in the enterococcal JH2-2 strain containing the necessary native biosynthetic machinery for enterocin production. The importance of these derivatives in AS-48 processing has been evaluated on the basis of the production and structural characterization of the corresponding derivatives. Notably, only two of them (Trp70Ala and Met1Ala derivatives) could be purified in different forms and amounts and are characterized for their bactericidal activity and secondary structure. We could not detect any production of AS-48 in JH2-2(pAM401-81(His-1Ile)) by using the conventional chromatographic techniques, despite the high efficiency of the culture conditions applied to produce this enterocin. Our results underline the different important roles of the mutated residues in (i) the elimination of the SP, (ii) the production levels and antibacterial activity of the mature proteins, and (iii) protein circularization. Moreover, our findings suggest that His-1 is critically involved in cleavage site recognition, its substitution being responsible for the blockage of processing, thereby hampering the production of the specific protein in the cellular culture supernatant.

Analysis of ColE1 MbeC unveils an extended ribbon-helix-helix family of nicking accessory proteins.

MbeC is a 13-kDa ColE1-encoded protein required for efficient mobilization of ColE1, a plasmid widely used in cloning vector technology. MbeC protein was purified and used for in vitro DNA binding, which showed that it binds specifically double-stranded DNA (dsDNA) containing the ColE1 oriT. Amino acid sequence comparison and secondary structure prediction imply that MbeC is related to the ribbon-helix-helix (RHH) protein family. Alignment with RHH members pointed to a conserved arginine (R13 in MbeC) that was mutated to alanine. The mutant MbeC(R13A) was unable to bind either single-stranded DNA or dsDNA. Limited proteolysis fragmented MbeC in two stable folding domains: the N-terminal domain, which contains the RHH motif, and the C-terminal domain, which comprises a signature shared by nicking accessory proteins. The results indicate that MbeC plays a similar role in conjugation as TraY and TrwA of plasmids F and R388, respectively. Thus, it appears that an extended, possibly universal mechanism of DNA conjugative processing exists, in which oriT-processing is carried out by relaxases assisted by homologous nicking accessory proteins. This mechanism seems to be shared by all major conjugative systems analyzed thus far.

Bent helix formation between RNA hairpins with complementary loops.

The initial interaction between the ColE1 plasmid specific transcripts RNA I and RNA II, which function as antisense regulators of plasmid replication, comprises a transient complex between complementary loops found within the RNA secondary structures. Multidimensional heteronuclear magnetic resonance spectroscopy was used to characterize complexes formed between model RNA hairpins having seven nucleotide complementary loops. Seven base pairs are formed in the loop-loop helix, with continuous helical stacking of the loop residues on the 3' side of their helical stems. A sharp bend in the loop-loop helix, documented by gel electrophoresis, narrows the major groove and allows bridging of the phosphodiester backbones across the major groove in order to close the hairpin loops at their 5'-ends. The bend is further enhanced by the binding of Rom, a ColE1 encoded protein that regulates replication.

[Analysis of bacterial colonization associated with Gigaspora margarita spores by green fluorescence protein (GFP) marked technology].

OBJECTIVE: We analyzed bacterial colonization associated with spores of arbuscular mycorrhizal fungi (AMF) Gigaspora margarita, to indicate their ecological niche, and to provide information for further researches on their populations or functions. METHODS: Six bacteria strains (Peanibacillus sp. M060106-1, Peanibacillus sp. M061122-2, Peanibacillus sp. M061122-6, Bacillus sp. M061122-4, Bacillus sp. M061122-10 and Brevibacillus sp. M061122-12) isolated from G. margarita spores were tagged with green fluorescence protein (GFP) using the carrier plasmid pNF8 (gfp-mut1). We analyzed the ecological niche and population dynamics of tagged strains on G. margarita under different conditions by using fluorescent microscope and/or plate counts. RESULTS: Four strains (M060106-1, M061122-6, M061122-10 and M061122-12) were tagged with GFP, showing high plasmid stability. These tagged strains possessed the basic characteristics identical to their original strains and, hence, were fit for short-term study of environmental colonization. All four GFP-tagged strains colonized the spore wall of G. margarita, and M061122-6 and M061122-12 further colonized the fungal hyphae. Under different pH conditions,the population dynamic of each GFP-tagged strain on the spores showed the same trend, i.e. first increased and then decreased, and the effects on the population size varied with different pH value. GFP-tagged strains colonized the spores of low viability more easily than those of high viability, and the population dynamic on the spores of high viability was different for each tagged strain. CONCLUSION: The isolated bacteria associated with G. margarita spores can re-colonize the fungal spores, whereas their colonizing ability depends on their characteristics and environmental factors. These data contributes to the further understanding of populations and functions of AMF-associated bacteria.

[Bifidobacterial plasmids and their application to genetic engineering].

Representatives of Bifidobacterium genus are considered to play many important roles in intestinal homeostasis. On the other hand, their molecular biology and genetics have been poorly studied. In order to broaden our understanding of their health-promoting mechanisms, it is extremely important to possess tools to manipulate them genetically. Another challenging task is to take advantage of genetic engineering technology for designing new probiotic bifidobacteria with unique therapeutic properties. An important step in such work is to isolate and characterize small bifidobacterial plasmids, which can be applied to the construction of cloning vectors. This article presents a review of several pioneering studied devoted to bifidobacterial plasmids and genetic engineering with bifidobacteria. Trends in and prospects of molecular genetics of bifidobacteria are discussed as well.

Control of ColE1 plasmid replication by antisense RNA.

One of the two major classes of regulatory strategies that control plasmid copy number involves recognition via base pairing between two plasmid-encoded complementary RNAs. The detailed analysis of this control circuitry has revealed some features of regulatory mechanisms based on RNA-RNA interaction that distinguish them from those based on protein-nucleic acid interaction. These features provide a framework with which to understand other regulatory mechanisms based on RNA-RNA interaction, and will aid in the design of efficient artificial antisense RNA systems.

A novel vector for positive selection of inserts harboring an open reading frame by translational coupling.

We have developed a novel vector pTCS, as a tool for efficient selection of open reading frame (ORF)-containing inserts. In pTCS clones containing an insert with an ORF a downstream marker gene (immE3, conferring resistance to colicin) is activated via translational coupling with the insert, and transformed cells can then be selected by exposure to colicin E3. Our method differs from previous methods in that the marker gene is activated without protein fusion, and that selection occurs irrespective of the reading frame of the insert.

Molecular and phenotypic profiling of sorbitol-fermenting Escherichia coli O157:H- human isolates from Finland.

This study investigated the occurrence of virulence-associated genes, including stx1, stx2, stx2c, stx2d, stx2e, eae and its subtypes (alpha, beta, gamma, epsilon), efa1, cdt-V cluster, enterohaemorrhagic Escherichia coli (EHEC)-hlyA, katP, espP, etpD, sfpA and the flagellar fliC gene, in nine sorbitol-fermenting (SF), beta-glucuronidase-positive E. coli O157:H- (non-motile) isolates obtained from humans in Finland between 1997 and 2001. In addition, the production of Shiga toxin (Stx), cytolethal distending toxin (CDT)-V and EHEC haemolysin (EHEC-Hly) was studied, and the phage type (PT) and pulsed-field gel electrophoresis (PFGE) types were determined. All nine isolates carried eae-gamma, efa1, EHEC-hlyA, etpD, sfpA and fliC; eight also harboured the cdt-V gene cluster and five were positive for stx2. None of the isolates harboured stx1, stx2c, stx2d, stx2e, katP or espP. All isolates harbouring the corresponding genes also produced Stx2 and CDT-V in titres ranging from 1:32 to 1:128 and from 1:2 to 1:4, respectively. None of the isolates expressed EHEC-Hly on enterohaemolysin agar. Seven isolates belonged to PT88 and two had a PT88 variant pattern. Seven isolates showed a close genetic relationship, with a PFGE similarity index (SI) of 92-98%. Two isolates, temporally the first and last, obtained 5 years apart, were the most divergent (SI of 71% and 85%, respectively). The study demonstrated that SF E. coli O157:H- isolates from Finland are closely related and show a close relationship with SF E. coli O157 strains isolated in Germany. This finding suggests a clonality of SF E. coli O157:H- isolates from different geographical regions.

Dramatic structural and thermodynamic consequences of repacking a protein's hydrophobic core.

BACKGROUND: Rop is an RNA binding, dimeric, four-helix bundle protein with a well-defined, regular hydrophobic core ideally suited for redesign studies. A family of Rop variants in which the hydrophobic core was systematically redesigned has previously been created and characterized. RESULTS: We present a structural and thermodynamic analysis of Ala2Ile2-6, a variant of Rop with an extensively redesigned hydrophobic core. The structure of Ala2Ile2-6 reveals a completely new fold formed by a conformational "flip" of the two protomers around the dimeric interface. The free-energy profile of Ala2Ile2-6 is also very different from that of wild-type Rop. Ala2Ile2-6 has a higher melting temperature than Rop, but undergoes a slightly smaller free-energy change on unfolding. CONCLUSIONS: The structure of Ala2Ile2-6, along with molecular modeling results, demonstrate the importance of tight packing of core residues and the adoption of favorable core side chain rotamer values in determining helix-helix interactions in the four-helix bundle fold. Structural disorder at the N and C termini of Ala2Ile2-6 provides a basis for the large differences in the enthalpy and entropy of Ala2Ile2-6 folding compared with wildtype Rop.

Assessment of quantitative models for plasmid ColE1 copy number control.

Two quantitative models of plasmid ColE1 copy number control are compared with respect to mathematical logic of derivation and application to experimental observations. Explanatory background material and clarifications are supplied for selected aspects of each model. Contrasting features are emphasized and experiments are suggested to distinguish between predictions of the models.

Analysis of establishment phase replication of the plasmid ColE1.

The replication regulatory mechanisms by which the small, multicopy plasmid ColE1 maintains a constant steady-state copy number have been extensively characterized by a combination of in vivo genetics and in vitro biochemistry. We have extended the analysis of replication control into the "establishment" phase of replication, when ColE1-directed replicons replicate more than once per cell generation and the intracellular concentrations of plasmid-encoded replication regulatory elements are changing. To study establishment phase replication, in which plasmid-directed replicons amplify from an initially low concentration to the characteristic, steady-state concentration, bacteriophage-plasmid hybrids, termed phasmids, were constructed. Phasmids were shown to exhibit stability, segregation, and incompatibility properties similar to those of the parent plasmid. Establishment phase replication was analyzed by measuring the number of phasmids per cell as a function of time after infection. We observed a linear increase in phasmid concentration until the steady-state concentration characteristic of the ColE1 plasmid component of the hybrid was reached. The number of cell doublings required for the phasmid concentration to reach steady-state was inversely related to cell growth rate. The observed amplification kinetics imply that the frequency of replication initiation per phasmid continually decreases until steady-state is reached. Kinetics of establishment phase amplification were sensitive to rate of expression of RNA II. A phasmid containing an up mutation in the RNA II promoter amplified at a 15-fold faster rate than the wild-type phasmid. Concentration of the ColE1 replication negative regulator (RNA I) was proportional to phasmid concentration throughout the amplification phase. These results suggest that the same elements that regulate steady-state replication also control establishment phase replication.

Premature termination of DNA replication in plasmids carrying two inversely oriented ColE1 origins.

In Escherichia coli plasmids carrying two inversely oriented ColE1 origins, DNA replication initiates at only one of the two potential origins. The other silent origin acts as a replication fork barrier. Whether this barrier is permanent or simply a pausing site remains unknown. Here, we used a repeated primer extension assay to map in vivo, at the nucleotide level, the 5' end of the nascent strand where initiation and blockage of replication forks occurs. Initiation occurred primarily at the previously defined origin, however, an alternative initiation site was detected 17 bp upstream. At the barrier, the lagging strand also terminated at the main initiation site. Therefore, the 5' end of the nascent strand at the barrier was identical to that generated during initiation. This observation strongly suggests that blockage of the replication fork at the silent origin is not just a pausing site but permanent, and leads to a premature termination event.

Mutations affecting RNA-DNA hybrid formation of the ColE1 replication primer RNA. Restoration of RNA I sensitivity to a copy-number mutant by second-site mutations.

Certain high copy-number mutants of the ColE1 plasmid produce a primer RNA that, unlike the wild-type, is resistant to inhibition by the plasmid-encoded replication inhibitor RNA I. We show that this resistance is associated with the ability of mutant primer RNA to hybridize to the DNA template strand more efficiently than does the wild-type transcript in vitro. We have isolated two second-site intramolecular suppressor mutations that partially restore wild-type copy number behavior to the high copy-number mutant in vivo. Each of these mutations alters a second base in primer RNA near the original mutation. We show that the primer RNA made by the pseudo-revertants regained wild-type-like sensitivity to RNA I in vitro. Also, the efficiency of RNA-DNA hybrid formation by the pseudo-revertant primer RNAs is restored to a level similar to that of wild-type primer. Using non-denaturing gel electrophoresis as an indication of RNA conformation, we identified two primer RNA conformers, each of 550 nucleotides, whose equilibrium distribution differs between wild-type and the mutant plasmid. The pseudo-revertant plasmids have a conformer distribution similar to that of wild-type, indicating that these primer sequence changes have long-range effects on primer conformation. An oligonucleotide complementary to the primer domain containing the mutation reduced hybrid formation when present during primer elongation. These results indicate that the copy-number behavior of these plasmids is a consequence of conformational alterations in primer RNA that alter its hybridization efficiency with the DNA template strand and its sensitivity to inhibition by RNA I.

Specificity determinants in interaction of the initiator (Rep) proteins with the origins in the plasmids ColE2-P9 and ColE3-CA38 identified by chimera analysis.

The ColE2-P9 rep protein specifically binds to the orgin and initiates DNA synthesis. Interaction of the Rep protein with the origins of plasmids ColE2-P9 and ColE-3-CA38 (one of the close relatives of ColE2-P9) is plasmid-specific. By using chimeric rep genes and chimeric origins we showed that the two region, A and B, in the C-terminal regions of the Rep proteins and the two sites alpha and beta, in the origins are important for the determination of specificity. When each of the A/alpha and B/beta pairs is from the same plasmids, the plasmid replication is efficient. On the other hand, if only the A/alpha pair is from the same plasmids, the plasmid replication is inefficient. For the region A, the plasmid-specificity is mainly determined by the presence or absence of a nine-amino acid sequence. For the region B, the specificity is probably determined by several amino acids. The region B, contains a segment of amino acid sequence which shows significant homology with the DNA recognition helices of various DNA binding proteins. At the site alpha, the single additional base-pair in the ColE3-CA38 origin can be either A/T or T/A. At the site beta, however, the single additional base-pair in the ColE2-P9 origin must be G/C. Among other possibilities we propose that the region A is a linker connecting the two domains in the Rep protein involved in DNA-binding and that the region B is a part of the sequence-specific DNA-binding domain.