To assess soil bacterial diversity, PCR systems consisting of several slightly different reverse primers together with forward primer F968-GC were used along with subsequent denaturing gradient gel electrophoresis (DGGE) or clone library analyses. In this study, a set of 13 previously used and novel reverse primers was tested with the canonical forward primer as to the DGGE fingerprints obtained from grassland soil. Analysis of these DGGE profiles by GelCompar showed that they all fell into two main clusters separated by a G/A alteration at position 14 in the reverse primer used. To assess differences between the dominant bacteria amplified, we then produced four (100-membered) 16S rRNA gene clone libraries by using reverse primers with either an A or a G at position 14, designated R1401-1a, R1401-1b, R1401-2a, and R1401-2b. Subsequent sequence analysis revealed that, on the basis of the about 410-bp sequence information, all four primers amplified similar, as well as different (including novel), bacterial groups from soil. Most of the clones fell into two main phyla, Firmicutes and Proteobacteria. Within Firmicutes, the majority of the clones belonged to the genus Bacillus. Within Proteobacteria, the majority of the clones fell into the alpha or gamma subgroup whereas a few were delta and beta proteobacteria. The other phyla found were Actinobacteria, Acidobacteria, Verrucomicrobia, Chloroflexi, Gemmatimonadetes, Chlorobi, Bacteroidetes, Chlamydiae, candidate division TM7, Ferribacter, Cyanobacteria, and Deinococcus. Statistical analysis of the data revealed that reverse primers R1401-1b and R1401-1a both produced libraries with the highest diversities yet amplified different types. Their concomitant use is recommended.Denaturing gradient gel electrophoresis (DGGE) is a powerful molecular technique in which DNA fragments of the same length but with different sequences can be separated (7,8,23,24,32). When applied to 16S rRNA genes, the method allows the dissection of microbial communities at the level of the phylogeny of their constituents. PCR applied to regions of this gene with conserved primers allows the generation of a mixture of amplicons which can be separated by DGGE. The technique was initially introduced into microbial ecology by Muyzer et al. in 1993 and has been widely used since its inception (21). Whereas Muyzer and coworkers originally proposed a system based on the V3 region of the 16S rRNA gene, Heuer et al. (12) and Smalla et al. (34) described a PCR-DGGE system based on the V6 region of this gene. This region has the highest variability within the whole rRNA gene, thus theoretically allowing for the most nearly optimal dissection of bacterial communities. Since its concoction, several different reverse primers for region V6-based DGGE have been described in the literature, some of which show sequence differences (6,15,16,25,26). Moreover, some of the reverse primers carrying the same name had different nucleotide compositions whereas, on the other hand, primers were found to have the same nu...

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The Laccaria proxima mycosphere strongly selects Variovorax paradoxus cells. Fifteen independent V. paradoxus strains, isolated from mycospheres sampled at two occasions, were investigated with respect to the occurrence of plasmids of sizes <60-100 kb. Two V. paradoxus strains, HB44 and BS64, were found to contain such plasmids, which were coined pHB44 and pBS64. Replicon typing using a suite of plasmid-specific PCR systems indicated that both plasmids belong to the IncP-1β group. Also, both were able to mobilize selectable IncQ group plasmids into Escherichia coli as well as Pseudomonas fluorescens. Moreover, they showed stable replication in these organisms, confirming their broad host range. Strain BS64 was cured of pBS64 and plasmid pHB44 was subsequently moved into this cured strain by making use of the IncQ group tracer plasmid pSUP104, which was then removed at elevated temperature. Thus, both plasmids could be screened for their ability to confer a phenotype upon strain BS64. No evidence for the presence of genes for xenobiotic degradation and/or antibiotic or heavy metal resistances was found for either of the two plasmids. Remarkably, both could stimulate the production of biofilm material by strain BS64. Also, the population densities of pBS64-containing strain BS64 were temporarily raised in liquid as well as soil systems (versus the plasmid-cured strain), both in the presence of the fungal host Lyophyllum sp. strain Karsten. Strikingly, plasmid pHB44 significantly enhanced the fitness of strain BS64 in soil containing Lyophyllum sp. strain Karsten, but decreased its fitness in soil supplemented with extra FeCl3. The effect was noted both in separate (no inter-strain competition) and joint (competition) inoculations.

The human body harbors numerous populations of microorganisms in various ecological niches. Some of these microbial niches, such as the human gut and the respiratory system, are well studied. One system that has been understudied is the urinary tract, primarily because it has been considered sterile in the absence of infection. Thanks to modern sequencing and enhanced culture techniques, it is now known that a urinary microbiota exists. The implication is that these species live as communities in the urinary tract, forming microbial ecosystems. However, the interactions between species in such an ecosystem remains unknown. Various studies in different parts of the human body have highlighted the ability of the pre-existing microbiota to alter the course of infection by impacting the pathogenicity of bacteria either directly or indirectly. For the urinary tract, the effect of the resident microbiota on uropathogens and the phenotypic microbial interactions is largely unknown. No studies have yet measured the response of uropathogens to the resident urinary bacteria. In this study, we investigate the interactions between uropathogens, isolated from elderly individuals suffering from UTIs, and bacteria isolated from the urinary tract of asymptomatic individuals using growth measurements in conditioned media. We observed that bacteria isolated from individuals with UTI-like symptoms and bacteria isolated from asymptomatic individuals can affect each other’s growth; for example, bacteria isolated from symptomatic individuals affect the growth of bacteria isolated from asymptomatic individuals more negatively than vice versa. Additionally, we show that Gram-positive bacteria alter the growth characteristics differently compared to Gram-negative bacteria. Our results are an early step in elucidating the role of microbial interactions in urinary microbial ecosystems that harbor both uropathogens and pre-existing microbiota.

Two novel plasmids, coined pHB44 and pBS64, were recently found in Variovorax paradoxus strains HB44 and BS64 isolated from the mycosphere of Laccaria proxima, on two different sampling occasions. We here describe the full sequences of pHB44 and pBS64 and establish their evolutionary placement and ecological function. Both plasmids, unique for mycospheric V. paradoxus, were around 58 kb in size. They possessed, in a very similar fashion, three main plasmid backbone regions, which were predicted to be involved in plasmid replication, central control of maintenance, and conjugational transfer. Phylogenetic inference on the basis of seven selected and concatenated plasmid backbone genes provided solid evidence for the placement of the two plasmids in the IncP-1β1 group, with the recently isolated IncP-1β1 plasmid pMBUI8 as the closest relative. A comparative analysis of the sequences present in each of the recombinational hot spots (RHS) I to III across plasmids pHB44, pBS64, and pMBUI8 revealed the insertions found in plasmids pHB44 and pBS64 to be different from those of pMBUI8. Whereas, in the former two plasmids, RHS I and III were devoid of any major inserts, their RHS II regions contained inserts of 15,043 (pHB44) and 16,406 kb (pBS64), against about 9,3 kb for pMBUI8. Interestingly, these regions were highly similar across plasmids pHB44 and pBS64, and differed from that of pMBUI8. Closer inspection revealed the insert in the former plasmids to contain, next to transposases, an “mmf” gene cassette previously reported to encode metal “responsiveness” in the PromA plasmid pMOL98. Whereas the plasmid pHB44 RHS II contained the canonical mmf sequence, that in pBS64 contained, in addition, a “two-gene duplicated region” flanking the mmf C2 gene. In vitro experiments on the growth and survival of strains with or without plasmid pHB44 suggested this plasmid was involved in the binding and import of Fe3+ as well as V3+ ions into the host cells, thus yielding a growth advantage under “metal ion-limiting” conditions. In addition, pHB44 was found to confer a bacitracin resistance phenotype to its host strain HB44. The metal import and bacitracin resistance traits were tentatively attributed to specific genes present in the RHS II inserts.