, 1990; Daims et al., 1999) were included (Table 1). Hybridization of P. riparius endosymbiont cells obtained from homogenized genitalia with Cy3-PAE444 resulted in intense

fluorescent labelling of all cells evaluated over the whole range of formamide concentrations from 0% to 80% (Fig. 1a). However, PAE444 hybridized nonspecifically to nontarget sequences of P. aeruginosa cells from 0% to 60% formamide (Fig. 1b). Further increase of formamide concentration (stringency) resulted in a significant loss of cells’ signal intensity. However, even the highest formamide concentration of 80% was not sufficient to cause dissociation of 50% of PAE444 high throughput screening compounds from nontarget cells of P. aeruginosa. Hybridization of Cy3-cPAE444 to P. riparius endosymbiont cells (Fig. 1a) resulted in equal fluorescent labelling as shown for Cy3-PAE444 in case of P. aeruginosa cells (Fig. 1b). Hybridization of a 1 : 1-mixture of Cy3-cPAE444 and unlabelled PAE444 to endosymbiont 16S rRNA gene was observed for the lowest applied formamide concentrations of 0% and 5% only. With concentrations >10%, the weak fluorescence-signal completely disappeared, indicating specific discrimination of P. aeruginosa cells vs. endosymbiont cells. Hybridization EPZ5676 nmr of P. aeruginosa cells with Cy3-cPAE444 resulted in 100% fluorescence intensity over the whole range of formamide concentrations from 0% to 80%.

Thus, the unlabelled oligonucleotide cPAE444 complementary to P. Fossariinae aeruginosa 16S rRNA gene was included as a competitor during hybridization to prevent the nonspecific hybridization of PAE444 with the nontarget sequence of P. aeruginosa. Hybridization of 1 : 1-mixed Cy3-PAE444 and unlabelled competitor probe cPAE444 to endosymbiont 16S rRNA gene resulted in intense fluorescent labelling of the cells hybridized with formamide concentrations from 0% to 80%, and nonspecific hybridization of Cy3-PAE444 to P. aeruginosa cells was not detectable at formamide

concentrations >20%. Thus, formamide concentration in the hybridization buffer was adjusted to 30% in all following hybridizations, and the concentrations of NaCl (X) and EDTA (Y) in the washing buffer were 112 and 5 mM, respectively. The data indicate that the hybridization protocol allows for a specific detection of Pseudomonas-like Paederus endosymbionts. The Pseudomonas-like Paederus endosymbionts were exclusively detected on a special layer entirely coating the egg shell in seven analysed section series of P. riparius eggs (Fig. 2a–d). Hundred percent of DAPI and Cy3-EUB-Mix-positive cells hybridized with Cy3-PAE444, indicating a ‘pure culture’ of endosymbionts (Fig. 2a–d). The interior of the investigated thin-sectioned eggs was always devoid of any bacteria as indicated by hybridization with Cy3-EUB-Mix (data not shown). Surface investigation of P. riparius eggs by SEM identified a granular layer, indicating microbial cells completely covering the eggshell (Fig. 3a and b).

Thus, a number of terms were required to describe problems related to the use of medications such as adverse drug reaction, adverse drug event, drug therapy problem and medication error. A further list of search terms was generated by referring to two key papers. The first article was a review on MRP classification systems by Van Mil et al.[24] which provided an overview and appraisal of classification

of medicine-related problems for use during the pharmaceutical care process and research in pharmacy. The second article by AbuRuz et al.[25] aimed to develop and validate a tool to classify and assess MRPs in which an MRP was referred to as ‘treatment related problem’. These two articles had also reported difficulties in identifying previous literature on MRPs learn more from databases. Each article suggested a list of search terms for ‘medicine-related problems’. The search terms reported by these articles include drug related selleck inhibitor problem,[24, 25] medicine related problem,[24, 25] drug therapy problem, treatment related problem,

therapy related problem, medication error and pharmaceutical care issue.[25] The different keywords used to search for relevant articles in this review are presented in Table 1. Drug related problem(s) OR Drug therapy problem(s) OR Drug self medication OR Drug self administration OR Drug toxicity OR Adverse drug reaction OR Drug interaction OR Drug intoxication OR drug contraindication OR Adverse drug effect OR Overdose OR Polypharmacy OR Drug evaluation OR Drug dose OR Drug monitoring OR Drug safety OR Drug screening OR Drug seeking behaviour OR Drug tolerability OR Drug tolerance OR Drug use OR Drug monitoring OR Drug utilisation OR Medicine related problem(s) OR Medication error(s) OR Medication adherence OR Medication compliance OR Medication therapy management OR Therapy related problem(s) OR Treatment related problem(s)

OR Pharmaceutical care issue(s) Ethnicity OR Ethnic group(s) OR Race OR Racial group(s) OR Religion OR Religious group(s) OR Minority group(s) United Kingdom OR Great Britain OR England A further difficulty was the limited reporting of the ethnic profile of participants in previous studies. It has been argued that the under-representation Methane monooxygenase of minority ethnic groups in studies may be because participants of ethnic minorities fail to understand the importance of the research process or they are unable to participate because of language barriers.[26] However, another possible explanation would be that some researchers have not received training or do not recognise the complexity or importance of incorporating the perspective of minority populations into their research and thus assume the cultural perspective or need of the majority in the conduct of their research.[27] The articles were selected through titles and abstracts by the first author of this paper (FA).

Autoinduction mediated by AHL signals has been well described in the plant pathogen P. stewartii ssp. stewartii (von Bodman et al., 2003) and has been reported recently in the pathogens P. agglomerans pv. gypsophilae and P. ananatis (Morohoshi et al., 2007; Chalupowicz et al., 2008). Based on the sequence homology to the pagRI genes of

P. agglomerans pv. gypsophilae (Chalupowicz et al., 2008; Rezzonico et al., 2009) the transcriptional regulator pagR and the AHL-synthase pagI genes (Pvag_pPag30141–Pvag_pPag30142) have been identified on plasmid VE-821 molecular weight pPag3. Using an A. tumefaciens biosensor (Shaw et al., 1997), AHL production was tested. For this purpose, the plant pathogenic strain P. ananatis LMG 2665 was added to the assay as a positive control. Pantoea vagans C9-1 has a positive autoinducer functional activity, but yields a weaker signal in the biosensor assay than P. ananatis LMG 2665 (Fig. 2). The variant P. vagans C9-1W lost this activity (Fig. 2), confirming that this strain selleck compound is not able to produce detectable AHLs.

Although the chromosome also contains a putative AHL synthase, located next to the sdiA gene encoding a LuxR-type transcriptional regulator (Lindsay & Ahmer, 2005; Smits et al., 2009), it can be concluded from the results of the biosensor assay that this chromosomal gene is not involved in the synthesis of the AHLs that can be detected with the A. tumefaciens biosensor. The PagRI quorum-sensing system plays a central role in the virulence of P. agglomerans pv. gypsophilae by regulating the expression of the T3SS (Chalupowicz et al., 2009). Its role in the ecological behavior of P. vagans and P. agglomerans strains that have functional pagRI genes is currently unknown (Rezzonico et al., 2009). The fact that most nonpathogenic strains lack a T3SS, however,

suggests that pagRI may have additional non-virulence-related functions in phytopathogenic pathovars. Siderophores are small molecules that bind Fe3+ with a high affinity and are synthesized by bacteria under iron starvation. The genome of P. vagans C9-1 contains biosynthetic genes for the catecholate siderophore enterobactin (ent-fep) PD184352 (CI-1040) and the hydroxamate siderophore desferrioxamine E (dfoJACS), which were reported to be produced by the strain (Feistner & Ishimaru, 1996). Siderophore biosynthesis can be an important biocontrol trait, as the strain may be able to compete with phytopathogens for the already limited supply of iron in planta. When spotted onto CAS siderophore indicator plates (Schwyn & Neilands, 1987), P. vagans C9-1 produces a large halo, indicative of siderophore synthesis, while variant C9-1W produces a small halo, just around the colony (Fig. 3). This difference can be attributed to the absence of the pPag3-encoded dfoJACS gene cluster (Pvag_pPag30339–Pvag_pPag30342), which confers the ability of desferrioxamine production to the strain. Pantoea vagans C9-1W was compared with the wild-type strain P.

Autoinduction mediated by AHL signals has been well described in the plant pathogen P. stewartii ssp. stewartii (von Bodman et al., 2003) and has been reported recently in the pathogens P. agglomerans pv. gypsophilae and P. ananatis (Morohoshi et al., 2007; Chalupowicz et al., 2008). Based on the sequence homology to the pagRI genes of

P. agglomerans pv. gypsophilae (Chalupowicz et al., 2008; Rezzonico et al., 2009) the transcriptional regulator pagR and the AHL-synthase pagI genes (Pvag_pPag30141–Pvag_pPag30142) have been identified on plasmid Wnt inhibitor pPag3. Using an A. tumefaciens biosensor (Shaw et al., 1997), AHL production was tested. For this purpose, the plant pathogenic strain P. ananatis LMG 2665 was added to the assay as a positive control. Pantoea vagans C9-1 has a positive autoinducer functional activity, but yields a weaker signal in the biosensor assay than P. ananatis LMG 2665 (Fig. 2). The variant P. vagans C9-1W lost this activity (Fig. 2), confirming that this strain JAK inhibitor is not able to produce detectable AHLs.

Although the chromosome also contains a putative AHL synthase, located next to the sdiA gene encoding a LuxR-type transcriptional regulator (Lindsay & Ahmer, 2005; Smits et al., 2009), it can be concluded from the results of the biosensor assay that this chromosomal gene is not involved in the synthesis of the AHLs that can be detected with the A. tumefaciens biosensor. The PagRI quorum-sensing system plays a central role in the virulence of P. agglomerans pv. gypsophilae by regulating the expression of the T3SS (Chalupowicz et al., 2009). Its role in the ecological behavior of P. vagans and P. agglomerans strains that have functional pagRI genes is currently unknown (Rezzonico et al., 2009). The fact that most nonpathogenic strains lack a T3SS, however,

suggests that pagRI may have additional non-virulence-related functions in phytopathogenic pathovars. Siderophores are small molecules that bind Fe3+ with a high affinity and are synthesized by bacteria under iron starvation. The genome of P. vagans C9-1 contains biosynthetic genes for the catecholate siderophore enterobactin (ent-fep) Fossariinae and the hydroxamate siderophore desferrioxamine E (dfoJACS), which were reported to be produced by the strain (Feistner & Ishimaru, 1996). Siderophore biosynthesis can be an important biocontrol trait, as the strain may be able to compete with phytopathogens for the already limited supply of iron in planta. When spotted onto CAS siderophore indicator plates (Schwyn & Neilands, 1987), P. vagans C9-1 produces a large halo, indicative of siderophore synthesis, while variant C9-1W produces a small halo, just around the colony (Fig. 3). This difference can be attributed to the absence of the pPag3-encoded dfoJACS gene cluster (Pvag_pPag30339–Pvag_pPag30342), which confers the ability of desferrioxamine production to the strain. Pantoea vagans C9-1W was compared with the wild-type strain P.

2 ± 8.3%, 91.5 ± 16.2%, and 87.8 ± 5.8%, respectively. When other competing substrates indicated in Fig. 3 were tested in the mutant, l-cystine was still transported into the cells despite the absence of the TcyABC system (data not shown), confirming the presence of other cystine transporters in S. mutans. Our results show that in addition to l-cystine transport, the TcyABC transporter participates in the uptake of l-cysteine, dl-cystathionine, l-djenkolic acid, and S-methyl-l-cysteine in S. mutans. The two transcriptional activators CysR and HomR are positive regulators

of the TcyABC and TcyDEFGH l-cystine transport systems, respectively (Sperandio et al., 2010). Our search of the selleck chemicals S. mutans UA159 genome revealed another putative LysR-type transcriptional regulator (LTTR) locus (SMU.2060) designated TcyR with homology (24%, 65/263) to the B. subtilis YtlI regulator. To determine the role of TcyR on the expression of the tcyABC operon, we constructed a TcyR insertion mutant (SmTcyR) and tested it under cystine starvation conditions. Gene expression was analyzed by quantitative real-time RT-PCR using cDNAs derived from S. mutans UA159 and mutant strains grown in modified MM with or without cystine. Relative to their expression in UA159,

the absence of TcyR resulted in an approximate 10.8-, 13.1-, and 5.2-fold induction of tcyA, BYL719 datasheet tcyB, and tcyC respectively, under cystine starvation relative to the cystine-fed state (Fig. 4). These results indicate that TcyR has a negative transcriptional role on the expression of tcyABC during cystine limited conditions. these LTTRs are generally positive regulators in prokaryotes (Leichert et al., 2003). Interestingly, the B. subtilis YtlI regulator with which the TcyR regulator shares homology is also a positive regulator. The negative regulator CymRSA in S. aureus (Coppee et al., 2001) showed no homology to our negative regulator TcyR. We also found that under cystine starvation, UA159 cells showed upregulation of the tcyABC and tcyR genes (Fig. 4). More specifically, the

tcysA, tcyB, and tcyC genes were upregulated by 3.3-, 2.4-, and 2.8-fold, whereas expression of tcyR was increased by c. 2.6-fold, thus suggesting induction of the transporter genes and its regulator under cystine-deprived nutritional conditions. This capability is likely advantageous under dense biofilm growth where conditions can become anaerobic and access to nutrients and free amino acids may be limited. In this environment, where cells are likely trying to scavenge cystine or cystine amino acid analogues, upregulation would be an advantage. To determine the effect of cystine starvation on S. mutans UA159 growth, wild-type and mutant strains were grown in MM medium devoid of cysteine–HCl, and growth was monitored using an automated optical density reader.

Majority of caries-active children had maxillary incisor caries, and the presence of dental caries in the maxillary incisors carried a high odds ratio for the child to have caries in the rest of the dentition. This caries pattern is not unique to this study check details and has been demonstrated in other studies[20, 21]. Alaluusua et al.[16] reported that visible plaque on the labial surfaces of maxillary incisors could predict the caries status of very young children (sensitivity: 83%; specificity: 92%). The results of

this study confirmed that an assessment of the presence of caries and the plaque accumulation of the 4 maxillary incisors may serve as an alternative to a full oral examination especially during public health epidemiology studies and be utilized by physicians and mid-level healthcare providers to detect caries in young children. At the time of the study, very few Singaporean children had been to a dentist. Furthermore, these children visited the dentist

only because they had dental decay requiring attention. Thus, this practice was not protective in the caries risk assessment, but rather appeared to be a consequence of the child having dental decay. In contrast PD0332991 in vivo to only 1% in Singapore, 37% of Hong Kong parents indicated that the first dental visit for their child should be around 1 year of age[22]. The American Academy of Pediatric Dentistry recommends that all children should have their first dental visit no later than 12 months of age[23]. Baricitinib Many Singaporean parents were unaware of the appropriate age for their child to have their first dental visit and felt that a visit to the dentist was warranted only if their child had tooth pain. Of those who reported an age, 5 years was thought by many parents to be an appropriate time for their child’s first dental visit in our study. Many parents cited

that their child did not require regular dental check-ups because they did not complain about their teeth. Homecare practices also appeared to be poor; close to 40% of children were brushing their teeth without supervision, a practice that is not aligned with the AAPD guidelines[24]. These worrisome attitudes and practices suggest that the establishment of a dental home at an early age was not a priority for Singaporean parents. Currently, the school dental health programme in Singapore provides free dental examination and treatment for school children (7 years of age and older), and this may have influenced parental perception on the appropriate age to visit the dentist. Additionally, there were no formalized public health dental services for toddlers and preschool children, which may explain the low awareness of the merits of preventive dental visits and subsequent utilization rate among preschool children.

, 2010). The disease symptoms include white or grey patches of filamentous mycelium on the body or the fins of freshwater fish. The cellular and molecular mechanisms underlying Saprolegnia

infection have not been studied extensively (Kamoun, 2003). Instead, considerably more is known about MK-2206 in vivo how plant pathogenic oomycetes infect their hosts. Most oomycetes generate asexual zoospores for dispersal, which encyst and germinate when they have reached a potential host. Saprolegnia parasitica is also able to generate both primary and secondary zoospores, whereby the latter type is infectious (Phillips et al., 2008; Bruno et al., 2010). Upon finding a host, some oomycetes form a swelling at the tip of a germ tube, called an appressorium, which forms a penetration peg to enter the host cell (Grenville-Briggs et al., 2008). These appressoria-like structures have not been described so far for S. parasitica. Biotrophic and hemibiotrophic plant pathogenic oomycetes can also generate specialized hyphal branches called haustoria. These are structures that invaginate Trametinib the plant cell and induce the formation of a plant-derived

extrahaustorial membrane with a gel-like layer between the extrahaustorial membrane and the haustorial wall, called the extrahaustorial matrix (Bushnell, 1972; Szabo & Bushnell, 2001). Within this extrahaustorial matrix, water and nutrients are exchanged between the pathogen and the host (Voegele & Mendgen, 2003). The extracellular space is also considered important for the trafficking of secreted proteins from the pathogen, including effector proteins (Ellis et al., 2006). Effector proteins are required to establish a successful infection, but if recognized, they can also trigger a host resistance response (Birch et al., 2006, 2009; Jones & Dangl, Selleckchem Decitabine 2006; Hogenhout et al., 2009). Some plant and animal pathogens have evolved intriguing molecular

mechanisms to inject or translocate potential effector proteins into their host cells (Coombes et al., 2004; Navarro et al., 2005; Birch et al., 2006; Jones & Dangl, 2006; Whisson et al., 2007). For example, bacterial pathogens can inject effector proteins into the host cytosol using a type-III secretion system, where these effectors can suppress basal/innate immunity, inhibit inflammatory responses, inhibit phagocytosis and induce apoptosis in macrophages (Hueck, 1998; Navarro et al., 2005; Galán & Wolf-Watz, 2006; Lewis et al., 2009). The eukaryotic parasite Plasmodium falciparum, the causative agent of malaria, is also able to translocate secreted proteins that contain a so-called PEXEL motif [amino acid motif RxLx (E, Q or D)] into the cytosol of red blood cells (Hiller et al., 2004; Marti et al., 2004; Hiss et al., 2008). During the blood stages of infection, the parasite invades mature human erythrocytes and develops within a parasitophorous vacuolar membrane.

, 2010). The disease symptoms include white or grey patches of filamentous mycelium on the body or the fins of freshwater fish. The cellular and molecular mechanisms underlying Saprolegnia

infection have not been studied extensively (Kamoun, 2003). Instead, considerably more is known about buy Apitolisib how plant pathogenic oomycetes infect their hosts. Most oomycetes generate asexual zoospores for dispersal, which encyst and germinate when they have reached a potential host. Saprolegnia parasitica is also able to generate both primary and secondary zoospores, whereby the latter type is infectious (Phillips et al., 2008; Bruno et al., 2010). Upon finding a host, some oomycetes form a swelling at the tip of a germ tube, called an appressorium, which forms a penetration peg to enter the host cell (Grenville-Briggs et al., 2008). These appressoria-like structures have not been described so far for S. parasitica. Biotrophic and hemibiotrophic plant pathogenic oomycetes can also generate specialized hyphal branches called haustoria. These are structures that invaginate FK866 datasheet the plant cell and induce the formation of a plant-derived

extrahaustorial membrane with a gel-like layer between the extrahaustorial membrane and the haustorial wall, called the extrahaustorial matrix (Bushnell, 1972; Szabo & Bushnell, 2001). Within this extrahaustorial matrix, water and nutrients are exchanged between the pathogen and the host (Voegele & Mendgen, 2003). The extracellular space is also considered important for the trafficking of secreted proteins from the pathogen, including effector proteins (Ellis et al., 2006). Effector proteins are required to establish a successful infection, but if recognized, they can also trigger a host resistance response (Birch et al., 2006, 2009; Jones & Dangl, NADPH-cytochrome-c2 reductase 2006; Hogenhout et al., 2009). Some plant and animal pathogens have evolved intriguing molecular

mechanisms to inject or translocate potential effector proteins into their host cells (Coombes et al., 2004; Navarro et al., 2005; Birch et al., 2006; Jones & Dangl, 2006; Whisson et al., 2007). For example, bacterial pathogens can inject effector proteins into the host cytosol using a type-III secretion system, where these effectors can suppress basal/innate immunity, inhibit inflammatory responses, inhibit phagocytosis and induce apoptosis in macrophages (Hueck, 1998; Navarro et al., 2005; Galán & Wolf-Watz, 2006; Lewis et al., 2009). The eukaryotic parasite Plasmodium falciparum, the causative agent of malaria, is also able to translocate secreted proteins that contain a so-called PEXEL motif [amino acid motif RxLx (E, Q or D)] into the cytosol of red blood cells (Hiller et al., 2004; Marti et al., 2004; Hiss et al., 2008). During the blood stages of infection, the parasite invades mature human erythrocytes and develops within a parasitophorous vacuolar membrane.

2 μm filtered distilled water. Also, 125 μL of 5% (w/v) phenol and 625 μL of ∼95% sulfuric acid were added simultaneously to the diluted samples in semi-micro photometer cuvettes (Plastibrand 759015). After 10 min incubation at room temperature and 15 min incubation at 30 °C, absorbance was measured at 490 nm against a glucose standard (0–100 μg mL−1). For bulk DNA measurements, 1 mL

aliquots of the homogenized biofilm-containing liquid cultures were lyzed by three freeze/thaw cycles (5 min at −20 °C and 5 min at 60 °C). DNA contents of the lyzate, and also DNA contents of the EPS extracts, were measured in 200 μL dilution series in black 96-well optical bottom microtiter plates (Nunc 265301). PI was added to a final concentration of 50 μM. Total fluorescence was measured check details on a Beckman–Coulter DTX880 multimode detector using a long-pass emission filter and 535 nm excitation light. Solutions of 0–100 μg mL−1 fish sperm DNA (Sigma 74782) were used as a standard. Nonpurgeable organic carbon contents were determined with a Total Organic Carbon Analyzer (TOC-Vwp; Shimadzu,

Columbia, MD) using a standard protocol. The sensitivity range of the method was 0.5–3500 mg L−1. Dynamic viscosities of cultures and extracts were determined by measuring elution times for draining 50 mL aliquots of the cultures or culture extracts through a glass burette (i.d.=2 mm). Using deionized water as a reference (=1.003 cSt), dynamic viscosities of the suspensions were determined Androgen Receptor Antagonist with =tC, with t as elution time and C as instrument constant. Exocellular β-glucosidase activity was measured according to Rath & Herndl, 1994. Replicate static cultures were mixed by vortexing, sampled, and supernatants were collected by centrifugation (10 000 g, 1 min) and microfiltration (0.2 μm). 4-Methylumbelliferyl β-d-glucopyranoside

was added (2.5 μM) and fluorescence was read after 30 min (265 nm excitation, 445-nm band pass emission; Cary Eclipse, Varian) against nonspiked control samples. Microfiltered sonication-lyzed P. putida cells were used to check method response. Idelalisib purchase Live/dead staining was performed as per an established protocol (Nielsen et al., 2009). In brief, replicate static cultures were mixed and two subsamples of 100 μL were removed; each of these was supplemented with 10 μL Sybr Green (Sybr Green I; Molecular Probes, Eugene, OR) from a 100 × stock solution, 10 μL PI (Invitrogen, Carlsbad, CA) from a 0.5 mg mL−1 stock solution, and 10 μL yellow–green fluospheres–carboxylate microspheres (F-8827; Molecular Probes) in a concentration of 2 × 107 mL−1 as internal standards for flow control. The samples were then supplemented with 870 μL MilliQ water containing 5 mM EDTA for outer-membrane permeabilization (Nebe-von-Caron et al., 2000). Samples were then vortexed, incubated in the dark for 15 min, and briefly vortexed again before being analyzed.

subtilis and Escherichia coli; however, the precise manner of SpoIISA toxicity remains unknown. In this work, we focused on the N-terminal, transmembrane domain of SpoIISA and verified the prediction of its topology. Using truncated SpoIISA constructs, we show that the entire transmembrane domain is required for its toxicity. Moreover, we propose that

the oligomerization of this transmembrane domain is crucial for activity of SpoIISA, possibly by forming a pore-like structure. “
“The pHW126-like plasmids are a recently discovered small group of cryptic plasmids replicating by the rolling circle mode. The replication origin of pHW126 consists of a conserved stretch, four perfect Ixazomib datasheet direct repeats and a so-called accessory region. The latter increases plasmid stability but is not absolutely necessary for replication. Here, we report that

deletion of the accessory region causes rapid multimerization of pHW126. While the number Afatinib in vitro of pHW126-units per cell remains constant, the number of physically independent plasmid molecules is reduced by approximately 40%, rendering random distribution to daughter cells less effective. A conserved inverted repeat within the accessory region could be identified as a sequence necessary for maintaining pHW126 in its monomeric form. A mutant version of pHW126 lacking this inverted repeat could be rescued by placing the single-strand initiation site (ssi) of pHW15 on the plus strand, while including the ssi in the opposite direction had no effect. Thus, our data provide evidence that multimer formation is, besides copy number

reduction and ssDNA accumulation, an additional means how loss of a mechanism ensuring efficient lagging strand synthesis may cause destabilization of rolling circle plasmids. Plasmids of bacteria appear in a wide variety of sizes, have different Vitamin B12 copy numbers and may encode various functions. Accordingly, plasmids have evolved different strategies for their maintenance. Huge circular plasmids usually replicate by the theta mechanism, are frequently self-transmissible and have a low copy number of just a few molecules per cell. Consequently, these plasmids depend on systems mediating partitioning, multimer resolution and postsegregational killing to ensure distribution to daughter cells. Small plasmids may also use the theta mode, but many of them replicate by the rolling circle mechanism or by strand displacement (del Solar et al., 1998; Rawlings & Tietze, 2001; Khan, 2005). Small plasmids are nonself-transmissible but may possess systems mediating mobilization in the presence of a conjugative plasmid (Francia et al., 2004; Garcillan-Barcia et al., 2009). Owing to their high copy number, small plasmids can rely on random distribution.