Using physical and genetic data, we have demonstrated that Rhizobium meliloti SU47 has a symbiotic megaplasmid, pRmeSU47b, in addition to the previously described nod-nif megaplasmid pRmeSU47a. This plasm'id includes four loci involved in exopolysaccharide (exo) synthesis as well as two loci involved in thiamine 1'iosynthesis. Mutations at the exo loci have previously been shown to result in the formation of nodules which lack infection threads (Inf)' and fail to fix nitrogen (Fix-). Thus, both megaplasmids contain genes involved in the formation of nitrogen-fixing root nodules. Mutations at two other exo loci were not located on either megaplasmid. To mobilize the megaplasmids, the oriT of plasmid RK2 was inserted into them. On alfalfa, Agrobacterium tumefaciens strains containing pRmeSU47a induced marked root hair curling with no infection threads and Fix-nodules, as reported by others. This plant phenotype was not observed to change with A. tumefaciens strains containing both pRmeSU47a and pRmeSU47b megaplasmids, and strains containing pRmeSU47b alone failed to curl root hairs or form nodules.A great many natural isolates of rhizobia, agrobacteria, and pseudomonads have been shown to carry a variety of large plasmids (for examples, see references 8, 21, and 38). Some of these are megaplasmids, with molecular masses over 450 megadaltons (38). Genes for a few functions have been localized to these large plasmids, notably including pathogenicity gepes for the Ti and Ri plasmids of agrobacteria (6, 21) and symbiotic nodulation (nod) and nitrogen fixation (nif) genes for the Sym megaplasmids of the fast-growing rhizobia (3,8,28,33,37,38). Nevertheless, the significance of this genomic organization remains obscure, although the fact that these are all plant-associated soil bacteria does suggest an underlying evolutionary cause.Rhizobial Sym megaplasmids are being characterized extensively with regard to symbiosis. In addition, Rhizobium meliloti 41 has recently been shown to carry a second megaplasmid, with a molecular weight very nearly that of pSym, on which a region for surface exclusion (although none for symbiotic functions) has tentatively been identified (2). A second megaplasmid has also been identified in R.

Enterobacter species were studied longitudinally in a children's hospital. In total, 287 Enterobacter isolates were obtained from 171 children in 15 different wards (from March 1995 through April 1997). Strains were typed by random amplified polymorphic DNA and pulsed-field gel electrophoresis, which were concordant in outcome. In total, 97 DNA types and 199 colonization events were identified. A predominant clone was isolated 111 times from 62 children; another clone was isolated 19 times from 10 patients. These clones caused 36% of all colonizations. In 34% of the children, Enterobacter clones were found in 2-4 patients. The remaining colonizations were due to unique Enterobacter isolates. A large proportion of the Enterobacter strains was acquired through cross-transmission. This finding contrasts with the prevailing opinion that resistant Enterobacter strains are selected primarily from the patient's own gut flora.

Transposon TnS encodes streptomycin resistance in addition to kanamycin-neomycin resistance. This resistance was not detectable in Escherichia coli but was efficiently expressed in Rhizobium meliloti and certain other strains. By analysis of cloned TnS restriction endonuclease fragments, the streptomycin resistance (str) gene was located in the right-hand side of the central region as the transposon is conventionally drawn. Transcription of str appeared to originate at pL, the promoter for the neo gene (neomycin phosphotransferase type II). Expression of streptomycin resistance in E. coli was obtained after cloning of the neo-str region downstream of a strong E. coli promoter. A construct in which PL was deleted also showed differential expression of streptomycin resistance. TnS is a transposon used widely for insertion mutagenesis in the genera Escherichia, Klebsiella, Pseudomonas, Agrobacterium, and Rhizobium (for reviews see references 3 and 27). Tn5 consists of a 2,700-base-pair (bp) core region flanked by two nearly identical copies of the insertion sequence IS50 in opposite orientations (4, 19). The right-hand inverted repeat (IS50R) codes for two proteins involved in both the transposition of TnS and the regulation of transposition (1, 17, 29). The left-hand inverted repeat (IS50L) carries an ochre mutation which results in the synthesis of two truncated proteins (1, 28, 29). In addition, this mutation appears to be in the promoter region for the neomycin phosphotransferase (neo) gene and results in ca. 10-fold increase in neomycin resistance in Escherichia coli over the ISSOR promoter sequence (28, 29). The 2,700-bp core region of TnS contains a 1,200-bp coding sequence for a neomycin phosphotransferase type II (2, 19) which confers resistance to the aminoglycoside antibiotics kanamycin and neomycin. There remains a 1,500bp region of DNA. TnS-mediated streptomycin resistance (Smr) has been reported by Mazodier et al. in Methylobacterium organophilum (21) and Forrai et al. in Rhizobium meliloti Rm4l (15). This Smr phenotype, however, is not expressed in E. coli. At about the same time, we detected this TnS Smr marker independently in R. meliloti SU47. In the course of characterizing the general R. meliloti transducing phage (M12 (13), we observed cotransduction of Nmr and Smr for several different TnS insertions, suggesting that the Smr was TnSencoded. We have confirmed this and localized a str gene at the right-hand side of TnS, in agreement with results of others published subsequently (22, 26; G. Selvaraj and V. N. Iyer, Abstr. 9th N. Am. Rhizobium Conf. 1983, P35, p. 18). In addition, we have found that TnS-encoded Smr can, in fact, be expressed in E. coli when str is cloned downstream from a stronger E. coli promoter, which suggests that the lack of Smr expression in E. coli is at the level of transcription. Bacterial strains and plasmids are listed in Table 1. Rich medium was LB (25), with 2.5 mM MgSO4 and 2.5 mM CaCI2 added for R. meliloti. Minimal medium was M9 (25) supplemented with 0.8% glucose a...