Arsenic mobilization and Fe(III) reduction in acetate-amended sediments collected from a range of depths from an aquifer with elevated groundwater arsenic concentrations in West Bengal were monitored over a 1 month period. Significant arsenic release was noted in sediment collected from 24 m and 45 m depth, with some Fe(III) reduction also observed in the 24 m sample. The structure of the microbial communities present in the sediments prior to incubation showed marked differences down the sediment column. Profiling of the microbial community in the 24 m and 45 m samples revealed a relatively complex make-up, with Acinetobacter species comprising the bulk of the 24 m sedimentary bacterial population, but no previously characterized As(V)-reducers were detected in either sample.

Our understanding of the distribution of integrons associated with multidrug resistance in Acinetobacter baumannii isolates around the world remains incomplete. The association between the class 1 and 2 integron A. baumannii-positive isolates (n = 60), recovered since 1982 from 11 Argentinean hospitals, and the circulating lineages, was investigated. While class 2 integrons were highly significantly associated with clonal lineage CC113B/CC79P (P = 0·009) and novel singletons (P = 0·001), class 1 integrons were found not to be associated with CC109B/CC1P or other lineages. The study reveals a differential distribution of class 2 integrons in lineages, and suggests that the prevalence of intI2 in Argentina is related to the emergence of novel singletons in recent years and to the abundance of CC113B/CC79P, which has been the local dominant lineage for several decades.

The effect of root-released compounds of transplastomic tobacco (Nicotiana tabacum) on the soil bacterial community structure, and their potential to support horizontal gene transfer (HGT) to bacteria have been studied. Soil microcosms were exposed to root-released compounds collected from transplastomic and non-transgenic tobacco cultivars. Cluster analysis of automated ribosomal intergenic spacer analysis (ARISA) profiles of the soil bacterial community after 48 h incubation grouped the transgenic cultivar apart from the non-transgenic, indicating that it had a rhizodeposition pattern different from the parental plants. However, these differences were less than between the two non-transgenic tobacco cultivars studied. NMR characterization of the root-released compounds showed some differences in chemical fingerprinting pattern between the transplastomic and the parental cultivar. However, the effect on bacterial community structure was transient, and tended to disappear after 96 h of incubation. The potential of root-released compounds as a source of transforming DNA for bacteria was investigated by using four potential recipient species. No transformants were obtained following exposure of all the recipients to the root-released compounds. Root-released compounds amended to transgene donor DNA decreased the transformation frequency of Acinetobacter baylyistrain ADP1200, while Azospirillum, Agrobacterium, and Sinorhizobium strains failed to develop competence also in the presence of an external added transgene source. Detection of plastid sequences by PCR suggested that a very low amount of fragmented plastid donor DNA was present in the root-released compounds.

Whereas the capability of DNA uptake has been well established for numerous species and strains of bacteria grown in vitro, the broader distribution of natural transformability within bacterial communities remains largely unexplored. Here, we investigate the ability of bacterial isolates from the gut of grass grub larvae (Costelytra zealandica (White); Coleoptera: Scarabaeidae) to develop natural genetic competence in vitro. A total of 37 mostly species-divergent strains isolated from the gut of grass grub larvae were selected for spontaneous rifampicin-resistance. Genomic DNA was subsequently isolated from the resistant strains and exposed to sensitive strains grown individually using established filter transformation protocols. DNA isolated from wild-type strains was used as a control. None of the 37 isolates tested exhibited a frequency of conversion to rifampicin-resistance in the presence of DNA at rates that were significantly higher than the rate of spontaneous mutation to rifampicin-resistance in the presence of wild-type DNA (the limit of detection was approximately < 1 culturable transformant per 109 exposed bacteria). To further examine if conditions were conducive to bacterial DNA uptake in the grass grubs gut, we employed the competent bacterium Acinetobacter baylyi strain BD413 as a recipient species for in vivo studies. However, no transformants could be detected above the detection limit of 1 transformant per 103 cells, possibly due to low population density and limited growth of A. baylyi cells in grass grub guts. PCR analysis indicated that chromosomal AcinetobacterDNA remains detectable by PCR for up to 3 days after direct inoculation into the alimentary tract of grass grub larvae. Nevertheless, neither transforming activity of the DNA recovered from the alimentary tract of grass grubs larvae nor competence of bacterial cells recovered from inoculated larvae could be shown.

The objective of this study was to evaluate the effect of insect larval feeding on the fate and genetic transformability of recombinant DNA from a transplastomic plant. Leaves of tobacco plants with an aadA antibiotic resistance gene inserted into their chloroplast genome were incubated with larvae of the tobacco hornworm Manduca sexta (Lepidoptera). The specifically designed Acinetobacter strain BD413 pBAB2 was chosen to analyze the functional integrity of the aadA transgene for natural transformation after gut passages. No gene transfer was detected after simultaneous feeding of leaves and the Acinetobacter BD413 pBAB2 as a recipient, even though 15% of ingested Acinetobacter BD413 cells could be recovered as viable cells from feces 6 h after feeding. Results with real-time PCR indicated that an average of 98.2 to 99.99% of the aadA gene was degraded during the gut passage, but the range in the number of aadA genes in feces of larvae fed with transplastomic leaves was enormous, varying from 5 × 106 to 1 × 109 copies.g-1. DNA extracted from feces of larvae fed with transplastomic leaves was still able to transform externally added competent Acinetobacter BD413 pBAB2in vitro. Transformation frequencies with concentrated feces DNA were in the same range as those found with leaves (10-4–10-6 transformants per recipient) or purified plasmid DNA (10-3–10-7). The presence of functionally intact DNA was also qualitatively observed after incubation of 30 mg freshly shed feces directly with competent Acinetobacter BD413 pBAB2, demonstrating that aadA genes in feces have a potential to undergo further horizontal gene transfer under environmental conditions.

The likelihood of gene transfer from transgenic plants to bacteria is dependent on gene number and the presence of homologous sequences. The large number of transgene copies in transplastomic (transgenes contained in the chloroplast genome) plant cells as well as the prokaryotic origin of the transgene, may thus significantly increase the likelihood of gene transfer to bacteria that colonize plant tissues. In order to assess the probability of such transfer, the length of homologous DNA sequences required between the transgene and the genome of the bacterial host was assessed. In addition, the probability that bacteria, which co-infect diseased plants, are transformable and have sequences similar to the flanking regions of the transgene was evaluated. Using Acinetobacter baylyi strain BD143 and transplastomic tobacco plants harboring the aadA gene (streptomycin and spectinomycin resistance), we found that sequences identical to the flanking regions containing as few as 55 nucleotides were sufficient for recombination to occur. Consequently, a collection of bacterial isolates able to colonize tobacco plant tissue infected by Ralstonia solanacearum strain K60 was obtained, screened for DNA sequence similarity with the chloroplastic genes accD and rbcL flanking the transgene, and tested for their ability to uptake extracellular DNA (broad host-range pBBR1MCS plasmids) by natural or electro-transformation. Results showed that among the 288 bacterial isolates tested, 8% presented DNA sequence similarity with one or both chloroplastic regions flanking the transgene. Two isolates, identified as Pseudomonas sp. and Acinetobacter sp., were able to integrate exogenous plasmid DNA by electro-transformation and natural transformation, respectively. Our data suggest that transplastomic plant DNA recipients might be present in soil bacterial communities.