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Class 1 and class 2 integrons in non-prevalent serovars of Salmonella enterica: structure and association with transposons and plasmids.

OBJECTIVES: To characterize class 1 and class 2 integrons which were simultaneously detected in non-typhoid Salmonella enterica strains of non-prevalent serovars, and to investigate their possible association with transposons and/or plasmids. METHODS: Eight multidrug-resistant S. enterica strains belonging to serovars Virchow (4), Panama (2), Grumpensis (1) and Worthington (1), each containing a class 1 and a class 2 integron, were analysed. Nested PCR amplification was used to determine the gene-cassette configuration of the integrons. Overlapping PCR amplifications were applied in integron-transposon linkage experiments. Conjugation and hybridization experiments were used to localize integrons and transposons in the bacterial genome (plasmid and chromosome associated). RESULTS: One of two different class 1 integrons (with variable regions of 1000 bp/aadA1 and 2300 bp/sat-smr-aadA1) inserted into Tn21-like transposons, were found to coexist with the class 2 integron (2300 bp/dfrA1-sat1-aadA1) of Tn7 in the analysed strains. Class 1 integrons were always found in large conjugative plasmids whereas apparently intact or defective copies of the Tn7 integron could be located on the same plasmid and/or the bacterial chromosome. CONCLUSIONS: This report describes different associations between mobile genetic elements that play a crucial role in the capture and spread of antimicrobial drug resistance. As far as we are aware, this is the first description of class 2 integrons in serovars Panama, Grumpensis and Worthington.

Chromosomes, Bacterial↗

Structural organization of the Corynebacterium glutamicum plasmid pCG100.

pCG100, a 3 kb cryptic plasmid of Corynebacterium glutamicum ATCC 13058, probably identical with pSR1 from C. glutamicum ATCC 19223, was characterized. The minimum region for autonomous replication was shown to be contained on a 1.9 kb BglII-NcoI fragment; a 380 bp HindIII-SphI fragment can replicate in the presence of the parental plasmid, which presumably provides a trans-acting replication factor. Derivatives of pCG100 are able to replicate in several Corynebacterium, Brevibacterium and Arthrobacter strains. pCG100 is compatible with pBL1, a cryptic plasmid of Brevibacterium lactofermentum. Shuttle plasmid vectors, containing the kanamycin-resistance gene from Tn903 or from Streptococcus faecalis as selectable markers and the AmpR, TetR or lacZ alpha genes for insertional inactivation, were constructed using the minimum replication fragment of pCG100.

Cloning, Molecular↗

Limited regions of homology between linear and circular plasmids encoding methylenomycin biosynthesis in two independently isolated streptomycetes.

pSV1 is a plasmid in Streptomyces violaceoruber SANK95570 that carries the methylenomycin biosynthetic (mmy) gene cluster. An ordered cosmid map and an EcoRI map have been constructed for pSV1, confirming that pSV1 is a 163 kb circular plasmid. The mmy gene cluster has been found on three different replicon structures; the circular plasmid pSV1, the 356 kb linear plasmid SCP1 and, via SCP1 integration, the linear chromosome of Streptomyces coelicolor A3(2). Comparison of pSV1 and SCP1 sequences revealed that the two plasmids have homology to each other only around the mmy and parAB regions, eliminating models in which pSV1 was generated by circularization of SCP1 or vice versa. It is likely that the mmy gene cluster was horizontally transferred as a set together with the parAB region in the comparatively recent evolutionary past.

Amino Acid Sequence↗

Structural and replicative diversity of large plasmids from sphingomonads that degrade polycyclic aromatic compounds and xenobiotics.

The plasmids from 16 sphingomonads which degrade various xenobiotics and polycyclic aromatic compounds were compared with the previously sequenced plasmid pNL1 from Sphingomonas aromaticivorans F199. The replicase genes repAaAb from plasmid pNL1 were amplified by PCR and used as a gene probe for the identification of plasmids belonging to the same incompatibility group as plasmid pNL1. Plasmids were prepared from various sphingomonads and hybridized with the repA gene probe. Positive hybridization signals were obtained with plasmids of approximately 160-195 kb from Sphingomonas subterranea and S. aromaticivorans B0695, which had been isolated from the same subsurface location as S. aromaticivorans F199. The repA probe also hybridized with plasmids from Sphingomonas xenophaga BN6, Sphingomonas sp. HH69 and Sphingomonas macrogoltabidus, which had been isolated from different continents and which utilize different organic compounds than S. aromaticivorans F199 and the other subsurface strains. The results of the hybridization experiments were confirmed by PCR experiments using primers deduced from the repAaAb region of plasmid pNL1. Nucleotide sequence comparisons suggested that three gene clusters were conserved between plasmid pNL1 and plasmid pBN6 from the naphthalenesulfonate- degrading strain S. xenophaga BN6. From these sequence comparisons, PCR primers were derived in order to detect the respective gene clusters in the other strains and to deduce their position relative to each other. These experiments demonstrated that all analysed subsurface strains harboured the same three gene clusters, but that the position and distance from each other of the clusters varied considerably among the different strains.

Biodegradation, Environmental↗

Asymbiotic acetylene reduction by a fast-growing cowpea Rhizobium strain with nitrogenase structural genes located on a symbiotic plasmid.

A procedure was designed which enabled the detection of ex planta nitrogenase activity in the fast-growing cowpea Rhizobium strain IHP100. Nitrogenase activity in agar culture under air occurred at a rate similar to that found for Bradyrhizobium strain CB756 but lower than that for Rhizobium strain ORS571. Hybridization studies showed that both nod and nif genes were located on a 410-kilobase Sym plasmid in strain IHP100.

Journal Article↗

Nitrogen-fixing nodules induced by Agrobacterium tumefaciens harboring Rhizobium phaseoli plasmids.

Rhizobium phaseoli CFN299 forms nitrogen-fixing nodules in Phaseolus vulgaris (bean) and in Leucaena esculenta. It has three plasmids of 185, 225, and 410 kilobases. The 410-kilobase plasmid contains the nitrogenase structural genes. We have transferred these plasmids to the plasmid-free strain Agrobacterium tumefaciens GMI9023. Transconjugants containing different combinations of the R. phaseoli plasmids were obtained, and they were exhaustively purified before nodulation was assayed. Only transconjugants harboring the 410-kilobase plasmid nodulate P. vulgaris and L. esculenta. Nodules formed by all such transconjugants are able to reduce acetylene. Transconjugants containing the whole set of plasmids from CFN299 nodulate better and fix more nitrogen than the transconjugants carrying only the Sym plasmid. Microscopic analysis of nodules induced by A. tumefaciens transconjugants reveals infected cells and vascular bundles. None of the A. tumefaciens transconjugants, not even the one with the whole set of plasmids from CFN299, behaves in symbiosis like the original R. phaseoli strain; the transconjugants produce fewer nodules and have lower acetylene reduction (25% as compared to the original R. phaseoli strain) and more amyloplasts per nodule. More than 2,000 bacterial isolates from nodules of P. vulgaris and L. esculenta formed by the transconjugants were analyzed by different criteria. Not a single rhizobium could be detected. Our results show that R. phaseoli plasmids may be expressed in the A. tumefaciens background and direct the formation of effective, differentiated nodules.

Conjugation, Genetic↗

Structural organization of pBC1, a cryptic plasmid from Bacillus coagulans.

The complete nucleotide sequence of the Bacillus coagulans plasmid pBC1 was determined. The sequence revealed an open reading frame encoding a polypeptide of 259 amino acids. This open reading frame shows sequence similarity to genes coding for replication-associated proteins in a group of gram-positive bacterial plasmids known to replicate via single-stranded intermediates. A region required for replication in cis, when the intact replicon is supplied in trans, was identified as well.

Amino Acid Sequence↗

Clarification of the structure of the ampicillin-resistance plasmid RSF0885 from Haemophilus influenzae HR-885 serotype b.

The nonconjugative ampicillin-resistance plasmid RSF0885 has been reported to be as small as 2.9 MDa and as large as 4.1 MDa with at least two restriction enzyme maps reported. In addition, the source of the original plasmid has been reported to be Haemophilus influenzae and Haemophilus parainfluenzae. Characterization of the source strains and sequencing data of the plasmids revealed that H. influenzae serotype b was the original source strain and that IS1-K in the larger plasmid was presumably acquired when the smaller plasmid was maintained in Escherichia coli in S. Falkow's laboratory during the late 1970s.

Ampicillin Resistance↗

[Integration of plasmids into E. coli K12 chromosomes, caused by a Bordetella transposon].

Transposition of Bordetella pertussis transposon in E. coli chromosome has been studied on a model of exclusion of donor multicopy pKK3 plasmid with coumermicin. TnBP3 induced the formation of co-integrates between the plasmid and chromosome. The structure of co-integrate was determined. Facts of exclusion of integrated structure and transposon transposition within integrated plasmid into new sites on a recipient chromosome were detected. Relationship between these processes and activity of bacterial cell recombination system has been determined.

Base Sequence↗

[Structural-functional organization of R-plasmid pBS222 with a broad range of bacterial hosts].

The broad host-range plasmid pBS222 is compatible with broad host-range plasmids of all known incompatibility groups and codes for tetracycline resistance. pBS222 is efficiently mobilized by Inc P-1 plasmid RP4 and is also capable of conjugal transfer with low efficiency to different gramnegative microorganisms. The size of the plasmid (17.2 Kb) has been determined and its physical map has been constructed. The plasmid harbours the unique sites for restriction endonucleases BglII, HindIII, HpaI, KpnI, SmaI and XbaI cleawage. The plasmid derivatives pBS352-pBS355 have been obtained that carry kan- and cam-determinants in addition to tet-gene. Plasmid pBS355 has been used to clone EcoRI-fragments of phage lambda DNA. The plasmid pBS222 regions essential for replication and maintenance have been localized by DNA hybridization analysis of its mini-derivatives pBS356 and 357. pBS222 is a convenient model for investigations of the plasmid replication and maintenance mechanisms in different bacterial hosts as well as for the construction of broad host-range vectors.

Conjugation, Genetic↗

[The role of phospholipids and their fatty acids in the structural organization of membranes of plasmid-containing and plasmid-less Salmonella derby cells].

The effect of R-plasmid on the qualitative and quantitative compositions of phospholipids and their fatty acids in bacterial (S. derby) cells has been studied. It has been found that the lack of R-plasmid in bacterial cells leads to a sharp decrease in the phospholipid content, in the phosphatidylcholine content in particular, and causes an increase in the per cent content of lysophosphatidylcholines. The per cent content of saturated and nonsaturated fatty acids of phospholipids in plasmid-free cells does not change appreciably. In the plasmid-free strain of S. derby the intensity of lipid peroxidation is associated with a relatively high content of alpha-tocopherol.

Cell Membrane↗

Small antibiotic resistance plasmids in Staphylococcus intermedius.

Antibiograms and plasmid profiles were evaluated for 116 Staphylococcus intermedius isolates collected from dogs in Germany and in the USA. Of the 26 S. intermedius isolates from Germany, 9 (34.6%) carried plasmids, while 20 (22.2%) of the 90 S. intermedius isolates from the USA were found to be plasmid-positive. Eight small resistance plasmids were identified and characterized using protoplast transformations and restriction endonuclease analyses. Five plasmids (3.8 and 3.9 kb) encoded for chloramphenicol resistance, 2 plasmids (each 2.5 kb) carried determinants for macrolide-lincosamide resistance, and one plasmid (4.5 kb) conferred resistance to tetracycline. Detailed restriction maps of these plasmids were constructed and served for structural comparisons with other small resistance plasmids found in staphylococci. These comparisons implied marked structural homologies with those prototype plasmids initially characterized in S. aureus of human origin.

Ampicillin Resistance↗

ColE1 plasmid replication: a simple kinetic description from a structured model.

Time constant analysis illustrates how a complex model for ColE-1-type plasmid replication can be reduced to a simple, realistic description of the overall replication kinetics. The analysis leads to the identification of the key parameters governing plasmid replication and to the estimation of parameters that are difficult to determine experimentally. The overall kinetics of ColE1-type plasmid replication are zerothorder in production and first order in dilution. Experimental data confirm our predictions. Similar analysis of the postulated mechanism for the action of the Rom protein reveals that it has no significant effect on plasmid replication dynamics.

Cell Cycle↗

Sequence analysis of plasmid pKJ50 from Bifidobacterium longum.

The complete nucleotide sequence of a plasmid, pKJ50, isolated from an intestinal bacterium, Bifidobacterium longum KJ, has been determined. The plasmid was analysed and found to be 4960 bp in size with a G+C content of 61.7 mol%. Computer analysis of sequence data revealed three major ORFs encoding putative proteins of 31.5 (ORFI), 24.5 (ORFII) and 38.6 kDa (ORFIII). ORFI encodes a protein with a pI of 10.18 and shows relatively high amino acid sequence similarity (more than 60%) with several plasmid replication proteins from Gram-positive and -negative bacteria. Southern blot analysis showed that pKJ50 accumulates an ssDNA intermediate, suggesting that it replicates by a rolling-circle mechanism. Upstream of ORFI, three sets of repeated sequences resembling iteron structures of related plasmids were identified. ORFIII encodes a protein with a pI of 10.97. It also shows a high level of amino acid sequence similarity with some plasmid mobilization proteins. Upstream of ORFIII, a 12 bp stretch resembles an oriT DNA sequence with inverted repeats identical to those found in conjugative plasmids. Hydropathy plot analysis of ORFII, encoding an acidic protein (pI = 4.95), suggests it is a transmembrane protein. Several interesting palindromic sequences, repeat sequences and hairpin-loop structures around ORFI, which might confer regulatory effects on the replication of the plasmid, were also noted. Reverse transcriptase PCR (RT-PCR) and in vitro translation confirmed the expression of ORFI and ORFII. RT-PCR produced amplified DNA fragments of the expected sizes, corresponding to ORFI and ORFII. However, no RT-PCR product corresponding to ORFIII was obtained. In vitro translation showed protein bands of the expected sizes, corresponding to each ORF. A shuttle vector capable of transforming Bifidobacterium animalis MB209 was constructed by cloning pKJ50 and a chloramphenicol resistance gene into pBR322.

Amino Acid Sequence↗

[Structuro-functional organization of segments of Co1A plasmid DNA, determining its stable inheritance].

Two par regions were localized within the structure of a small colicinogenic plasmid ColA. One of them functions at the expense of plasmid multimere resolution. Analysis of the nucleotide sequence of the region revealed the existence of essential homology with the par locus of plasmid ColE1. As compared to E. coli C600, the function of multimere forms' resolution of plasmid DNA in E. coli C is reduced or absent due to par regions of the ColE1 type. Par regions of various degrees of homology with the par locus of ColE1 were localized by Southern hybridization within the structure of colicinogenic plasmids ColN and ColD. The stabilization of the colicinogenic plasmids is believed to be also determined by the functioning of genes connected with the synthesis and action of colicin.

Bacteriocin Plasmids↗

Structure and function in Escherichia coli of plasmids containing pyrimidine/purine-biased stretch originated from the 5'-flanking region of the basidiomycete ras gene.

The Basidiomycete ras gene possesses a pyrimidine-rich stretch (CT-motif) with a short (7 bases) mirror repeat in which its major transcription start point is contained. To analyze the tertiary structure induced by the CT/AG-biased sequence and its effect on gene expression in supercoiled plasmids in Escherichia coli, the DNA fragment containing the ras CT/AG sequence was inserted into the EcoRI site on pBR322 in both orientations and the resulting pBR322 derivatives, named pBR-CT[ras] and pBR-invCT[ras] were introduced into E. coli strains DM800 (deltatopA gyrB225) and JM109 (topA+ gyrA96). In pBR-CT [ras] the pyrimidine-rich sequence is on the pBR322 tetracycline-resistance gene (tet)coding strand and in pBR-invCT[ras] the complementary purine-rich sequence is on this strand. DNAs of pBR-CT[ras] and pBR-invCT[ras] isolated from DM800 were frequently cleaved with single-strand-specific S1 nuclease within the CT/AG sequence, showing the formation of extended open structure. Compared with those carrying pBR322, DM800 and JM109 carrying pBR-CT [ras] showed much higher levels of tetracycline resistance (Tcr), while both strains carrying pBR-invCT[ras] showed clearly lower levels of Tcr. pBR-CT [ras] and pBR-invCT [ras], however, conferred reduced activity of beta-lactamase on DM800 and JM109. pBR-CT [ras] derivatives lacking the counterpart of the mirror repeat did not form the S1-cleavable open structure within the CT/AG sequence and conferred pBR322-like Tcr and beta-lactamase activity. The tertiary structure formed in the CT/AG sequence via the mirror repeat was suggested to affect the expressions of pBR322-tet and -bla genes.

Base Sequence↗