Conjugative pili of IncP plasmids, and the Ti plasmid T pilus are composed of cyclic subunits.
(33/3280)
TrbC propilin is the precursor of the pilin subunit TrbC of IncP conjugative pili in Escherichia coli. Likewise, its homologue, VirB2 propilin, is processed into T pilin of the Ti plasmid T pilus in Agrobacterium tumefaciens. TrbC and VirB2 propilin are truncated post-translationally at the N terminus by the removal of a 36/47-residue leader peptide, respectively. TrbC propilin undergoes a second processing step by the removal of 27 residues at the C terminus by host-encoded functions followed by the excision of four additional C-terminal residues by a plasmid-borne serine protease. The final product TrbC of 78 residues is cyclized via an intramolecular covalent head-to-tail peptide bond. The T pilin does not undergo additional truncation but is likewise cyclized. The circular structures of these pilins, as verified by mass spectrometry, represent novel primary configurations that conform and assemble into the conjugative apparatus. (+info)
Molecular characterization of the SHV-11 beta-lactamase of Shigella dysenteriae.
(34/3280)
A beta-lactamase with an M(r) of 29,000 and a pI of 7.6 was partially purified from a clinical isolate of Shigella dysenteriae. The bla gene encoded the SHV-11 enzyme carrying the substitution Leu-->Gln at position 35 and was linked to a strong promoter. This variant, unlike the prototype SHV-1 enzyme, hydrolyzed oxacillin, cloxacillin, and oxyiminocephalosporins such as cefotaxime. (+info)
UV stimulation of chromosomal marker exchange in Sulfolobus acidocaldarius: implications for DNA repair, conjugation and homologous recombination at extremely high temperatures.
(35/3280)
The hyperthermophilic archaeon Sulfolobus acidocaldarius exchanges and recombines chromosomal markers by a conjugational mechanism, and the overall yield of recombinants is greatly increased by previous exposure to UV light. This stimulation was studied in an effort to clarify its mechanism and that of marker exchange itself. A variety of experiments failed to identify a significant effect of UV irradiation on the frequency of cell pairing, indicating that subsequent steps are primarily affected, i.e., transfer of DNA between cells or homologous recombination. The UV-induced stimulation decayed rather quickly in parental cells during preincubation at 75 degrees, and the rate of decay depended on the incubation temperature. Preincubation at 75 degrees decreased the yield of recombinants neither from unirradiated parental cells nor from parental suspensions subsequently irradiated. We interpret these results as evidence that marker exchange is stimulated by recombinogenic DNA lesions formed as intermediates in the process of repairing UV photoproducts in the S. acidocaldarius chromosome. (+info)
Essential components of the Ti plasmid trb system, a type IV macromolecular transporter.
(36/3280)
The trb operon from pTiC58 is one of three loci that are required for conjugal transfer of this Ti plasmid. The operon, which probably codes for the mating bridge responsible for pair formation and DNA transfer, contains 12 genes, 11 of which are related to genes from other members of the type IV secretion system family. The 12th gene, traI, codes for production of Agrobacterium autoinducer (AAI). Insertion mutations were constructed in each of the 12 genes, contained on a full-length clone of the trb region, using antibiotic resistance cassettes or a newly constructed transposon. This transposon, called mini-Tn5Ptrb, was designed to express genes downstream of the insertion site from a promoter regulated by TraR and AAI. Each mutation could trans complement downstream Tn3HoHo1 insertions in the trb operon of full-sized Ti plasmids. When marker-exchanged into the transfer-constitutive Ti plasmid pTiC58DeltaaccR mutations in trbB, -C, -D, -E, -L, -F, -G, and -H abolished conjugal transfer from strain UIA5, which lacks the 450-kb catabolic plasmid pAtC58. However, these mutants retained residual conjugal transfer activity when tested in strain NT1, which contains this large plasmid. The trbJ mutant failed to transfer at a detectable frequency from either strain, while the trbI mutant transferred at very low but detectable levels from both donors. Only the trbK mutant was unaffected in conjugal transfer from either donor. Transfer of each of the marker-exchange mutants was restored by a clone expressing only the wild-type allele of the corresponding mutant trb gene. An insertion mutation in traI abolished the production of AAI and also conjugal transfer. This defect was restored by culturing the mutant donor in the presence of AAI. We conclude that all of the trb genes except trbI and trbK are essential for conjugal transfer of pTiC58. We also conclude that mutations in any one of the trb genes except traI and trbJ can be complemented by functions coded for by pAtC58. (+info)
Functional insights into pGI2, a cryptic rolling-circle replicating plasmid from Bacillus thuringiensis.
(37/3280)
Detailed functional analysis revealed the modular organization of pGI2, a 9672 bp plasmid from Bacillus thuringiensis H1.1 that harbours the 4149 bp transposon Tn4430. Whereas the pGI2 leading-strand replicon was identified through deletion experiments, sequence comparisons indicated the presence of an ssot-like single-strand origin commonly found among Bacillus plasmids. Southern hybridization confirmed the existence of ssDNA intermediates, but only in the case of plasmid derivatives lacking the ssot site. Moreover, the pGI2 replication protein Rep displayed significant similarity with that of pTX14-3, a 7-6 kb plasmid from B. thuringiensis serovar israelensis, suggesting that both elements are representatives of a new family of rolling-circle replicating (RCR) plasmids. In addition, both plasmids share a conserved 320 bp region downstream of their rep genes which, in the case of pGI2, appeared indispensable for replication. This region is therefore likely to correspond to, or to be part of, the actual double-strand origin of both plasmids. Another interesting feature of pGI2 is the presence of a mobilization (Mob) protein, as demonstrated by its ability to be mobilized by the conjugative plasmid pAW63 from B. thuringiensis serovar kurstaki HD73. The same transfer system was also used to unambiguously demonstrate similar properties of the related Mob-like protein from pTX14-3. A closer analysis of this family of related Mob proteins suggested a subdomanial organization among its members. Finally, the 270 residue pGI2 ORF2 was shown to be related to ORF43 of pMRC01, a 60 kb conjugative plasmid from Lactococcus lactis subsp. lactis. Although no function has been assigned to the putative ORF43 protein, it is located downstream of a bacteriocin operon, next to an IS946 element. pGI2 appears thus far as an assemblage of functional modules with no obvious metabolic function, presumably acting as a reservoir of carrier (rep and sso), rearrangement (Tn4430) or recruiting (Mob) entities for its bacterial host. (+info)
A variety of gram-positive bacteria carry mobile mef genes.
(38/3280)
The mefE gene codes for a membrane bound efflux protein, which confers resistance to macrolides, and has been identified in Streptococcus pneumoniae. A variety of gram-positive organisms were examined. Twenty-six isolates of S. pneumoniae carried mefE and were resistant to erythromycin (MIC of 2-16 mg/L). Two additional isolates of Emr S. pneumoniae carried both ermB and mefE(MIC of 16-128 mg/L). One Micrococcus luteus, one Corynebacterium jeikeium, three Corynebacterium spp., two viridans streptococci and seven Enterocccus spp. also carried mef genes. It was possible to move the mef gene from all 11 S. pneumoniae tested to susceptible S. pneumoniae and/or Enterococcus faecalis recipients. The addition of DNase (1 g/L) did not affect the gene transfer. It was also possible to move the mef gene from donor Enterococcus spp., viridans streptococci, M. luteus, C. jeikeium and Corynebacterium spp. to E. faecalis recipients. Transconjugant isolates were resistant to erythromycin (MIC = 16 mg/L). Hybridization with a labelled mef oligonucleotide probe against Southern blots and bacterial dot blots confirmed the presence of the mef genes. This is the first time that a mobile mef gene has been identified in four different genera, from three distinct geographical locations. (+info)
A comparison of the kinetics of plasmid transfer in the conjugation systems encoded by the F plasmid from Escherichia coli and plasmid pCF10 from Enterococcus faecalis.
(39/3280)
Quantitative measurements of horizontal DNA transfer are critical if one wishes to address questions relating to ecology, evolution and the safe use of recombinant bacteria. Traditionally, the efficiency of a conjugation system has been described by its transfer frequency. However, transfer frequencies can be determined in many ways and may be sensitive to physical, chemical and biological conditions. In this study the authors have used the mechanistic similarity between bacterial conjugation and simple enzyme catalysis in order to calculate the maximal conjugation rate (Vmax) and the recipient concentration (K(m)) at which the conjugation rate is half its maximal value, for two different conjugation systems: the F plasmid from Escherichia coli and plasmid pCF10 from Enterococcus faecalis. The results are compared with the data obtained from the aggregation-mediated conjugation system encoded on pXO16 from Bacillus thuringiensis. The conjugation systems analysed are fundamentally different; however, they have some characteristics in common: they are able to sustain conjugative transfer in liquid medium and the transfer efficiencies are very high. Conjugation encoded by the F plasmid in E. coli involves the formation of small aggregates (2-20 cells), established by sex pili, and the plasmid's maximal conjugation rate was estimated to be approximately 0.15 transconjugants per donor per minute. Pheromone-induced conjugation in Ent. faecalis, which involves the formation of large aggregates, was found to proceed at a maximal conjugation rate of 0.29 transconjugants per donor per minute. Also, the K(m) value differed significantly between these conjugation systems; this may reflect the inherent differences in mating pair formation and transfer mechanisms. In these conjugation systems, the donors underwent a 'recovery period' between rounds of conjugative transfer and newly formed transconjugants required a period of about 40-80 min to mature into proficient donors. (+info)
Comparison of proteins involved in pilus synthesis and mating pair stabilization from the related plasmids F and R100-1: insights into the mechanism of conjugation.
(40/3280)
F and R100-1 are closely related, derepressed, conjugative plasmids from the IncFI and IncFII incompatibility groups, respectively. Heteroduplex mapping and genetic analyses have revealed that the transfer regions are extremely similar between the two plasmids. Plasmid specificity can occur at the level of relaxosome formation, regulation, and surface exclusion between the two transfer systems. There are also differences in pilus serology, pilus-specific phage sensitivity, and requirements for OmpA and lipopolysaccharide components in the recipient cell. These phenotypic differences were exploited in this study to yield new information about the mechanism of pilus synthesis, mating pair stabilization, and surface and/or entry exclusion, which are collectively involved in mating pair formation (Mpf). The sequence of the remainder of the transfer region of R100-1 (trbA to traS) has been completed, and the complete sequence is compared to that of F. The differences between the two transfer regions include insertions and deletions, gene duplications, and mosaicism within genes, although the genes essential for Mpf are conserved in both plasmids. F+ cells carrying defined mutations in each of the Mpf genes were complemented with the homologous genes from R100-1. Our results indicate that the specificity in recipient cell recognition and entry exclusion are mediated by TraN and TraG, respectively, and not by the pilus. (+info)