Conjugative plasmid transfer in gram-positive bacteria.

Conjugative transfer of bacterial plasmids is the most efficient way of horizontal gene spread, and it is therefore considered one of the major reasons for the increase in the number of bacteria exhibiting multiple-antibiotic resistance. Thus, conjugation and spread of antibiotic resistance represents a severe problem in antibiotic treatment, especially of immunosuppressed patients and in intensive care units. While conjugation in gram-negative bacteria has been studied in great detail over the last decades, the transfer mechanisms of antibiotic resistance plasmids in gram-positive bacteria remained obscure. In the last few years, the entire nucleotide sequences of several large conjugative plasmids from gram-positive bacteria have been determined. Sequence analyses and data bank comparisons of their putative transfer (tra) regions have revealed significant similarities to tra regions of plasmids from gram-negative bacteria with regard to the respective DNA relaxases and their targets, the origins of transfer (oriT), and putative nucleoside triphosphatases NTP-ases with homologies to type IV secretion systems. In contrast, a single gene encoding a septal DNA translocator protein is involved in plasmid transfer between micelle-forming streptomycetes. Based on these clues, we propose the existence of two fundamentally different plasmid-mediated conjugative mechanisms in gram-positive microorganisms, namely, the mechanism taking place in unicellular gram-positive bacteria, which is functionally similar to that in gram-negative bacteria, and a second type that occurs in multicellular gram-positive bacteria, which seems to be characterized by double-stranded DNA transfer.     

Conjugative Plasmid Transfer in Gram-Positive Bacteria

Conjugative transfer of bacterial plasmids is the most efficient way of horizontal gene spread, and it is therefore considered one of the major reasons for the increase in the number of bacteria exhibiting multiple-antibiotic resistance. Thus, conjugation and spread of antibiotic resistance represents a severe problem in antibiotic treatment, especially of immunosuppressed patients and in intensive care units. While conjugation in gram-negative bacteria has been studied in great detail over the last decades, the transfer mechanisms of antibiotic resistance plasmids in gram-positive bacteria remained obscure. In the last few years, the entire nucleotide sequences of several large conjugative plasmids from gram-positive bacteria have been determined. Sequence analyses and data bank comparisons of their putative transfer (tra) regions have revealed significant similarities to tra regions of plasmids from gram-negative bacteria with regard to the respective DNA relaxases and their targets, the origins of transfer (oriT), and putative nucleoside triphosphatases NTP-ases with homologies to type IV secretion systems. In contrast, a single gene encoding a septal DNA translocator protein is involved in plasmid transfer between micelle-forming streptomycetes. Based on these clues, we propose the existence of two fundamentally different plasmid-mediated conjugative mechanisms in gram-positive microorganisms, namely, the mechanism taking place in unicellular gram-positive bacteria, which is functionally similar to that in gram-negative bacteria, and a second type that occurs in multicellular gram-positive bacteria, which seems to be characterized by double-stranded DNA transfer.     

Conjugative transposons: an unusual and diverse set of integrated gene transfer elements.

Conjugative transposons are integrated DNA elements that excise themselves to form a covalently closed circular intermediate. This circular intermediate can either reintegrate in the same cell (intracellular transposition) or transfer by conjugation to a recipient and integrate into the recipient's genome (intercellular transposition). Conjugative transposons were first found in gram-positive cocci but are now known to be present in a variety of gram-positive and gram-negative bacteria also. Conjugative transposons have a surprisingly broad host range, and they probably contribute as much as plasmids to the spread of antibiotic resistance genes in some genera of disease-causing bacteria. Resistance genes need not be carried on the conjugative transposon to be transferred. Many conjugative transposons can mobilize coresident plasmids, and the Bacteroides conjugative transposons can even excise and mobilize unlinked integrated elements. The Bacteroides conjugative transposons are also unusual in that their transfer activities are regulated by tetracycline via a complex regulatory network.     

Transfer of plasmid RSF1010 by conjugation from Escherichia coli to Streptomyces lividans and Mycobacterium smegmatis.

The plasmid RSF1010 belongs to a class of plasmids (IncQ) that replicate in a range of bacterial hosts. Although non-self-transmissible, it can be mobilized at high frequency between different gram-negative bacterial species if transfer functions are supplied in trans. We report the transfer of RSF1010 by conjugation from Escherichia coli to the gram-positive actinomycetes Streptomyces lividans and Mycobacterium smegmatis. In its new hosts, the plasmid was stable with respect to structure and inheritance and conferred high-level resistance to streptomycin and sulfonamide. This is the first reported case of conjugative transfer of a naturally occurring plasmid between gram-negative and gram-positive bacteria.     

Cultivation-independent examination of horizontal transfer and host range of an IncP-1 plasmid among gram-positive and gram-negative bacteria indigenous to the barley rhizosphere

The host range and transfer frequency of an IncP-1 plasmid (pKJK10) among indigenous bacteria in the barley rhizosphere was investigated. A new flow cytometry-based cultivation-independent method for enumeration and sorting of transconjugants for subsequent 16S rRNA gene classification was used. Indigenous transconjugant rhizosphere bacteria were collected by fluorescence-activated cell sorting and identified by cloning and sequencing of 16S rRNA genes from the sorted cells. The host range of the pKJK10 plasmid was exceptionally broad, as it included not only bacteria belonging to the alpha, beta, and gamma subclasses of the Proteobacteria, but also Arthrobacter sp., a gram-positive member of the Actinobacteria. The transfer frequency (transconjugants per donor) from the Pseudomonas putida donor to the indigenous bacteria was 7.03 x 10(-2) +/- 3.84 x 10(-2). This is the first direct documentation of conjugal transfer between gram-negative donor and gram-positive recipient bacteria in situ.     

Transformation of the gram-positive bacterium Clavibacter xyli subsp. cynodontis by electroporation with plasmids from the IncP incompatibility group.

We report the transformation of a gram-positive bacterium, Clavibacter xyli subsp. cynodontis, with several plasmids in the IncP incompatibility group from gram-negative bacteria. Our results suggest that IncP plasmids may be transferable to other gram-positive organisms. After optimizing electroporation parameters, we obtained a maximum of 2 x 10(5) transformants per microgram of DNA. The availability of a transformation system for this bacteria will facilitate its use in indirectly expressing beneficial traits in plants.     

Mobilization function of the pBHR1 plasmid, a derivative of the broad-host-range plasmid pBBR1

The pBHR1 plasmid is a derivative of the small (2.6-kb), mobilizable broad-host-range plasmid pBBR1, which was isolated from the gram-negative bacterium Bordetella bronchiseptica (R. Antoine and C. Locht, Mol. Microbiol. 6:1785-1799, 1992). Plasmid pBBR1 consists of two functional cassettes and presents sequence similarities with the transfer origins of several plasmids and mobilizable transposons from gram-positive bacteria. We show that the Mob protein specifically recognizes a 52-bp sequence which contains, in addition to the transfer origin, the promoter of the mob gene. We demonstrate that this gene is autoregulated. The binding of the Mob protein to the 52-bp sequence could thus allow the formation of a protein-DNA complex with a double function: relaxosome formation and mob gene regulation. We show that the Mob protein is a relaxase, and we located the nic site position in vitro. After sequence alignment, the position of the nic site of pBBR1 corresponds with those of the nick sites of the Bacteroides mobilizable transposon Tn4555 and the streptococcal plasmid pMV158. The oriT of the latter is characteristic of a family of mobilizable plasmids that are found in gram-positive bacteria and that replicate by the rolling-circle mechanism. Plasmid pBBR1 thus appears to be a new member of this group, even though it resides in gram-negative bacteria and does not replicate via a rolling-circle mechanism. In addition, we identified two amino acids of the Mob protein necessary for its activity, and we discuss their involvement in the mobilization mechanism.     

Efficient transfer of conjugative plasmids by multipoint inoculation and some observations on host range and prevalence of R plasmids.

The conjugational transfer of R plasmids was demonstrated using a simple manually operated multipoint inoculator apparatus (MIP) allowing rapid inoculation and later dilution and plating of 25 mating mixtures simultaneously. Forty-five R plasmids belonging to groups F, I, N, and others originally recovered in Escherichia coli K-12 were studied in this as well as in other hosts. The semiquantitative MIP conjugation method was more efficient than conventional matings, particularly when performed in two steps employing E. coli K-12 as intermediate host. Both as donor and as recipient, E. coli K-12 was the most “suitable” general host of the set of plamids studied, although with many plasmids the degree of expression of their transfer functions varied with the host. The expression of fertility in parental bacteria as well as factors in the new host not studied appeared to be of greater importance for the conjugational transfer of a plasmid than the host-specified restriction of plasmid deoxyribonucleic acid by the recipient strain. The MIP conjugation method was successfully used also during screening for transferable R plasmids in gram-negative bacteria present in urine and fecal specimens of humans. The use of a restrictionless mutant instead of a restricting K-12 recipient enabled the detection of additional plasmids. The labor and media-saving MIP conjugation method thus also offers efficiency and is very practical for the performance of large numbers of plasmid matings, for example, in studies of compatibilty, host range, and mobilization of plasmids, as well as for screening purposes.     

Analysis of the etiological structure and dynamics of antibiotic resistance in the causative agents of suppurative inflammatory diseases in the patients at a large general hospital

The structure of the causative agents isolated from patients with pyoinflammatory infections in 1980-1983 was analysed. It was shown that the surgical and urological infections were mainly caused by gram-negative bacteria. The other pyoinflammatory infections were mainly due to gram-positive cocci. A relatively high frequency of the strains of gram-negative bacteria, especially among Pseudomonas spp. and Enterobacter spp., resistant to aminoglycoside antibiotics, such as gentamicin, sisomycin and tobramycin with preserved sensitivity to amikacin and netilmicin in the majority of the strains was shown. Among the beta-lactam antibiotics cephotaxim and cephalotin were most active against gram-negative bacteria and staphylococci, respectively. The majority of the antibiotic resistant strains of gram-negative bacteria had analogous structures and levels of resistance to 7-12 antibiotics which might indicate the occurrence of 1-2 resistance plasmids among the clinical strains.     

Plasmid-encoded resistance to arsenic and antimony.

Resistance determinants to the toxic oxyanionic salts of arsenic and antimony are found on plasmids of both gram-negative and gram-positive organisms. In most cases these provide resistance to both the oxyanions of +III oxidation state, antimonite and arsenite, and the +V oxidation state, arsenate. In both gram-positive and -negative bacteria, resistance is correlated with efflux of the anions from cells. The determinant from the plasmid R773, isolated from a gram-negative organism, has been studied in detail. It encodes an oxyanion-translocating ATPase with three subunits, a catalytic subunit, the ArsA protein, a membrane subunit, the ArsB subunit, and a specificity factor, the ArsC protein. The first two form a membrane-bound complex with arsenite-stimulated ATPase activity. The determinants from gram-positive bacteria have only the arsB and arsC genes and encode an efflux system without the participation of an ArsA homologue.     

Polypeptide antibiotic 26a from Bacillus subtilis. III. Physicochemical and biological in vitro properties.

Antibiotic 26a, a weakly basic (pK1 3.85 and pK2 7.1) polypeptide compound, has been recovered from the fermentation fluids of bacillus subtilis cultures as hydrochloride salt easily soluble in water and dimethylsulphoxide, sparingly soluble in lower alcohols and insoluble in several organic solvents. At low concentrations 26a was effective against gram-positive bacteria, mainly micrococci and corynebacteria, moderately active against mycobacteria, and inactive against gram-negative bacteria, yeasts and moulds even at 300 microgram/ml concentration. From the viewpoint of elemental analysis, electrometric titration, optical rotation, UV, IR and NMR spectra, amino acid composition, molecular weight and biological observations, 26a can be considered as an antibiotic, if not identical, then closely related to bacitracin family polypeptides.     

Production of antibacterial substances by resident corynebacteria isolated from human skin

Coryneform bacteria, especially lipophilic species, form stable but not dominant population on a human skin. This position is probably controlled by secretion of bacteriocin-like substances, which act directly on coexisting bacteria. Among 118 investigated corynebacteria belonging to seven species/taxa and isolated from human skin, 90% possessed an ability to produce such substances. The spectrum of their activity was restricted to killing gram-positive bacteria, but along with corynebacteria it also covered cocci, with Staphylococcus aureus in this group. This feature was revealed better on low pH media (pH 5.6) and media with 1.5% NaCl for cocci, but on pH 7,4 for corynebacteria.     

Are gram‐positive bacteria capable of electron transfer across their cell wall without an externally available electron shuttle?

The extensive contributions by Terry Beveridge to our understanding of the differences in cell wall organization with respect to structure, chemistry and compartmentalization between gram-positive and gram-negative bacteria are summarized. These contributions greatly aided in conceptualization of recent discoveries concerning electron export and import across cell walls of some gram-negative bacteria. Although electron export and import across the cell wall by any gram-positive has not been documented so far, Beveridge's observations and concepts concerning cell walls of gram-positive bacteria suggest potential mechanisms by which such electron transfer may occur.     

Enterococcus faecalis Gene Transfer under Natural Conditions in Municipal Sewage Water Treatment Plants

The ability of Enterococcus faecalis to transfer various genetic elements under natural conditions was tested in two municipal sewage water treatment plants. Experiments in activated sludge basins of the plants were performed in a microcosm which allowed us to work under sterile conditions; experiments in anoxic sludge digestors were performed in dialysis bags. We used the following naturally occurring genetic elements: pAD1 and pIP1017 (two so-called sex pheromone plasmids with restricted host ranges, which are transferred at high rates under laboratory conditions); pIP501 (a resistance plasmid possessing a broad host range for gram-positive bacteria, which is transferred at low rates under laboratory conditions); and Tn916 (a conjugative transposon which is transferred under laboratory conditions at low rates to gram-positive bacteria and at very low rates to gram-negative bacteria). The transfer rate between different strains of E. faecalis under natural conditions was, compared to that under laboratory conditions, at least 10⁵-fold lower for the sex pheromone plasmids, at least 100-fold lower for pIP501, and at least 10-fold lower for Tn916. In no case was transfer from E. faecalis to another bacterial species detected. By determining the dependence of transfer rates for pIP1017 on bacterial concentration and extrapolating to actual concentrations in the sewage water treatment plant, we calculated that the maximum number of transfer events for the sex pheromone plasmids between different strains of E. faecalis in the municipal sewage water treatment plant of the city of Regensburg ranged from 10⁵ to 10⁸ events per 4 h, indicating that gene transfer should take place under natural conditions.     

Facile recovery of individual high-molecular-weight, low-copy-number natural plasmids for genomic sequencing

Sequencing of the large (>50 kb), low-copy-number (<5 per cell) plasmids that mediate horizontal gene transfer has been hindered by the difficulty and expense of isolating DNA from individual plasmids of this class. We report here that a kit method previously devised for purification of bacterial artificial chromosomes (BACs) can be adapted for effective preparation of individual plasmids up to 220 kb from wild gram-negative and gram-positive bacteria. Individual plasmid DNA recovered from less than 10 ml of Escherichia coli, Staphylococcus, and Corynebacterium cultures was of sufficient quantity and quality for construction of high-coverage libraries, as shown by sequencing five native plasmids ranging in size from 30 kb to 94 kb. We also report recommendations for vector screening to optimize plasmid sequence assembly, preliminary annotation of novel plasmid genomes, and insights on mobile genetic element biology derived from these sequences. Adaptation of this BAC method for large plasmid isolation removes one major technical hurdle to expanding our knowledge of the natural plasmid gene pool.     

Comparative biology of IncQ and IncQ-like plasmids.

Plasmids belonging to Escherichia coli incompatibility group Q are relatively small (approximately 5 to 15 kb) and able to replicate in a remarkably broad range of bacterial hosts. These include gram-positive bacteria such as Brevibacterium and Mycobacterium and gram-negative bacteria such as Agrobacterium, Desulfovibrio, and cyanobacteria. These plasmids are mobilized by several self-transmissible plasmids into an even more diverse range of organisms including yeasts, plants, and animal cells. IncQ plasmids are thus highly promiscuous. Recently, several IncQ-like plasmids have been isolated from bacteria found in environments as diverse as piggery manure and highly acidic commercial mineral biooxidation plants. These IncQ-like plasmids belong to different incompatibility groups but have similar broad-host-range replicons and mobilization properties to the IncQ plasmids. This review covers the ecology, classification, and evolution of IncQ and IncQ-like plasmids.     

Mobilization transfer of the pUB110 plasmid between gram-positive bacteria]

The three factor crosses between the donor strain Bacillus subtilis 168 harbouring the plasmid pUB102-4, Bacillus thuringiensis strain carrying the mobilizing plasmid pAM beta 1 and recipient strain Lactobacillus fermenti were conducted in order to elaborate the optimal conditions of the plasmid pUB102-4 mobilization for transfer into gram-positive microorganisms and to elucidate the possible expression of endogluconase genes in a lactobacillus strain. The Lactobacillus fermenti transconjugants carrying the pUB102-4 plasmid were obtained in the three factor reciprocal crosses with the streptococcus recipient strain and Bacillus subtilis recipients. The presence of the plasmids in transconjugants was confirmed by colony hybridization with the [32P]-labelled plasmid DNA and KMC-ase activity in transconjugant cells. The proposed system of crosses using the high copy number plasmid derivatives of pUB110 mobilized with high frequency by the pAM beta 1 plasmid demonstrates the possibility to increase the circle of gram-positive host bacteria avoiding time and labour consuming operations.     

Applications of fluorophore-containing microbial growth media.

Media containing the fluorogenic compound 8-anilino-1-naphthalene sulfonic acid may be used to discriminate between gram-positive and gram-negative bacteria and to differentiate between various species of bacteria. Fluorescent light emitted from colonies of gram-negative bacteria on 8-anilino-1-naphthalene sulfonic acid-containing agar was visually more intense than that on gram-positive bacteria. The emitted light from the gram-negative bacteria differed in wave-lengths from that of light emitted by colonies of gram-positive bacteria. The fluorescent intensity of colonies on complete 8-anilino-1-napthalene sulfonic acid agar supplemented with 1% of single substrates varied depending on the bacterial species, thus allowing the development of profiles used to identify 12 different species.