Martin J. Day

Elevated abundance of bacteriophage infecting bacteria in soil.

ABSTRACT Here we report the first direct counts of soil bacteriophage and show that substantial populations of these viruses exist in soil (grand mean = 1.5 × 107 g−1), at least 350-fold more than the highest numbers estimated from traditional viable plaque counts. Adding pure cultures of a Serratia phage to soil showed that the direct counting methods with electron microscopy developed here underestimated the added phage populations by at least eightfold. So, assuming natural phages were similarly underestimated, virus numbers in soil averaged 1.5 × 108 g−1, which is equivalent to 4% of the total population of bacteria. This high abundance was to some extent confirmed by hybridizing colonies grown on Serratia and Pseudomonas selective media with cocktails of phage infecting these bacteria. This showed that 8.9 and 3.9%, respectively, hybridized with colonies from the two media and confirmed the presence of phage DNA sequences in the cultivable fraction of the natural population. Thus, soil phage, like their aquatic counterparts, are likely to be important in controlling bacterial populations and mediating gene transfer in soil.

Influence of plant developmental stage on microbial community structure and activity in the rhizosphere of three field crops

Seasonal shifts in rhizosphere microbial populations were investigated to follow the influence of plant developmental stage. A field study of indigenous microbial rhizosphere communities was undertaken on pea (Pisum satvium var. quincy), wheat (Triticum aestivum var. pena wawa) and sugar beet (Beta vulgaris var. amythyst). Rhizosphere community diversity and substrate utilization patterns were followed throughout a growing season, by culturing, rRNA gene density gradient gel electrophoresis and BIOLOG. Culturable bacterial and fungal rhizosphere community densities were stable in pea and wheat rhizospheres, with dynamic shifts observed in the sugar beet rhizosphere. Successional shifts in bacterial and fungal diversity as plants mature demonstrated that different plants select and define their own functional rhizosphere communities. Assessment of metabolic activity and resource utilization by bacterial community-level physiological profiling demonstrated greater similarities between different plant species rhizosphere communities at the same than at different developmental stages. Marked temporal shifts in diversity and relative activity were observed in rhizosphere bacterial communities with developmental stage for all plant species studied. Shifts in the diversity of fungal and bacterial communities were more pronounced in maturing pea and sugar beet plants. This detailed study demonstrates that plant species select for specialized microbial communities that change in response to plant growth and plant inputs.

Plasmid Transfer between Strains of Pseudomonas aeruginosa on Membrane Filters Attached to River Stones

A naturally occurring mercury-resistance, conjugative plasmid, designated pQM1, was isolated from a bacterial population on the surface of stones from a river using Pseudomonas aeruginosa as a recipient. This was a narrow-host-range plasmid [IncP-13; 165 MDa; Tra+, Hgr, fluorescein mercuric acetater, merbrominr, Phi(E79), UVr] confined to some Pseudomonas spp. It was used to demonstrate transfer between bacteria on stones in laboratory microcosm experiments and in situ. Transfer occurred (3.3 X 10(-1) to 6.8 X 10(-9) per recipient) at all the temperatures used (6-20 degrees C), although frequencies were lower in the cold. Nutrient status also affected transfer frequency, rich conditions promoting transfer. The presence of competing bacteria in the natural epilithon lowered transfer frequencies, but when unscrubbed stones were heat treated, transfer was enhanced, perhaps because of nutrient release from the heated epilithon.

Bacterial genetics in natural environments

Section I: General Aspects of Studying Genetics in Nature.- 1 Genetic approaches to the study of gene transfer in microbial communities.- 2 Factors influencing the dissemination of DNA by bacterial conjugation.- 3 Factors limiting gene transfer in bacteria.- 4 Phage genetics and ecology.- Section II: Aquatic Habitats.- 5 Plasmid transfer in the epilithon.- 6 Laboratory standardised biofilms as a tool to investigate genetic transfers in water systems.- 7 Survival of laboratory and freshwater bacteria carrying an extrachromosomal xylE gene in freshwater microcosms.- 8 Gene transfer in marine environments.- 9 Gene transfer in activated sludge.- Section III: Terrestial Habitats.- 10 Plasmid transfer between soil bacteria.- 11 Gene transfer in polluted soils.- 12 The potential for gene exchange between rhizosphere bacteria.- 13 The use of a Sesbania rostrata microcosm for studying gene transfer among microorganisms.- 14 Plasmid transfer to indigenous bacteria in soil and rhizosphere: problems and perspectives.- 15 Use of wide host range promoters to monitor the fate of recombinant DNA in soil.- 16 The role of soil bacteria in risk assessment analysis.- 17 Gene transfer between streptomycetes in soil.- 18 The survival of genetically engineered microorganisms and bacteria on inanimate surfaces and in animals.- Section IV: Conclusions.- 19 Plasmid transfer and the release of genetically engineered bacteria in nature: a discussion and summary.

In situ transfer of an exogenously isolated plasmid between Pseudomonas spp. in sugar beet rhizosphere

Summary: A method for studying plasmid transfer in the rhizosphere is described. This work demonstrates plasmid transfer in an unenclosed rhizosphere under field conditions. The donor (Pseudomonas marginalis 376N) and recipient (Pseudomonas aureofaciens 381R) bacteria and the conjugative mercury resistance plasmid (pQBR11) studied were all isolated from the bacterial community indigenous to sugar beet rhizosphere. Spontaneous nalidixic acid and rifampicin resistant mutants of these bacteria were used as donors and recipients of pQBR11 for in situ matings. Fresh field soil was mixed with donors and recipients to give a soil mating mix (SMM) which was placed underground on the surface of a sugar beet root storage organ. Plasmid transfer in the SMM was determined after 24 h at frequencies between 5·1 × 10−5 and 1·3 × 10−8 transconjugants per recipient. Higher transfer frequencies (1·3 × 10−2 to 1·7 × 10−6) were recorded on the peel adjacent to the SMM. No transfer of mercury resistance was detected in SMM controls incubated at 20°C in vitro or placed in soil at distances of more than 5 cm from plants.

Seasonal Population Dynamics and Interactions of Competing Bacteriophages and Their Host in the Rhizosphere

ABSTRACT We describe two prolonged bacteriophage blooms within sugar beet rhizospheres ensuing from an artificial increase in numbers of an indigenous soil bacterium. Further, we provide evidence of in situ competition between these phages. This is the first in situ demonstration of such microbial interactions in soil. To achieve this, sugar beet seeds were inoculated with Serratia liquefaciensCP6RS or its lysogen, CP6RS-ly-Φ1. These were sown, along with uninoculated seeds, in 36 field plots arranged in a randomized Latin square. The plots were then sampled regularly over 194 days, and the plants were assayed for the released bacteria and any infectious phages. Both the lysogen and nonlysogen forms of CP6RS survived equally well in situ, contradicting earlier work suggesting lysogens have a competitive disadvantage in nature. A Podoviridae phage, identified as ΦCP6-4, flourished on the nonlysogen-inoculated plants in contrast to those plants inoculated with the lysogen. Conversely, the Siphoviridae phage ΦCP6-1 (used to construct the released lysogen) was isolated abundantly from the lysogen-treated plants but almost never on the nonlysogen-inoculated plants. The uninoculated plants also harbored some ΦCP6-1 phage up to day 137, yet hardly any ΦCP6-4 phages were found, and this was consistent with previous years. We show that the different temporal and spatial distributions of these two physiologically distinct phages can be explained by application of optimal foraging theory to phage ecology. This is the first time that such in situ evidence has been provided in support of this theoretical model.

Factors Affecting Conjugal Transfer of Plasmids Encoding Mercury Resistance from Pure Cultures and Mixed Natural Suspensions of Epilithic Bacteria

Sixty-five pure cultures of epilithic bacteria were examined for their ability to transfer mercury resistance to Pseudomonas aeruginosa; five isolates transferred plasmids encoding mercury resistance with frequencies ranging from 8.4 x 10(-8) to 2.8 x 10(-3) per recipient. Two of the plasmids, pQM3 and pQM4, encoded narrow-spectrum mercury resistance, pQM3 also encoded streptomycin resistance, and both plasmids were broad host range. Maximum transfer frequencies of epilithic plasmids from pure cultures occurred over the range 10-25 degrees C at 3.5 g C l-1 and with donor to recipient ratios of 0.4-30. Transfer occurred over a range of pH values (pH 5.0-8.0) but the effect of pH was most significant at non-optimal temperature. Anaerobiosis inhibited transfer of one epilithic plasmid, pQM1, but not that of pQM3. Plasmids encoding mercury resistance were also transferred from mixed natural suspensions of epilithic bacteria (MNS) to Pseudomonas spp. on agar in the laboratory. Transfer from MNS occurred over a wide range of environmentally relevant conditions with maximum frequencies (2 x 10(-5) per recipient) after 24 h, at 25 degrees C, pH 5.5-8.0 and on a medium containing 10 g C l-1. The optimal initial cell density of MNS and recipient was 1.7 x 10(5) c.f.u. cm-2 and highest frequencies were obtained with donor to recipient ratios ranging from 1.2 x 10(-1) to 1.7 x 10(-3). Most of the plasmids (54%) from MNS transferred from their original P. aeruginosa transconjugants to a Pseudomonas putida strain, with frequencies ranging from 1.1 x 10(-6) to greater than 1.0 x 10(-1) per recipient. The majority (80%) of the plasmids were larger than 300 kb and all of these large plasmids encoded UV resistance in addition to mercury resistance. Twenty-one plasmids greater than 300 kb were analysed by restriction digests and were shown to be similar, with only minor structural alterations. One of these alterations was associated with the acquisition of streptomycin resistance. Overall, these results suggest that the epilithic bacteria examined possess the potential to transfer mercury resistance within the epilithon under a wide range of environmentally relevant conditions.

Plasmid transfer in the epilithon

Plasmids have been commonly found in bacteria from a variety of natural habitats. In estuarine water and sediment from Chesapeake Bay 46% of heterotrophs contained plasmids,1 whilst in a South Wales river sediment only 10–15% possessed these replicons2. It is probable that many of these plasmids are conjugative as Burton et al.2 found 86% were larger than 30 kb. Furthermore, Jobling et al3 found that 25% of the mercury resistant bacteria from the River Mersey transferred plasmid encoded, mercury resistance to Escherichia coli. It is also likely that most Gram negative genera are potential recipients for at least some of these naturally occurring plasmids. Support for this comes from a recent study in which the broad host range, self transmissible plasmid R68 was found to transfer from Pseudomonas aeruginosa into 38% of the freshly isolated, heterotrophic bacteria tested.4 The genera which accepted the plasmid were Pseudomonas, Acinetobacter, Alcaligenes, Chromobacter- ium, Achromobacter and Moraxella, which are all very common aquatic bacteria.5

Characterization of Serratia isolates from soil, ecological implications and transfer of Serratia proteamaculans subsp. quinovora Grimont et al. 1983 to Serratia quinivorans corrig., sp. nov.

Eleven strains of Serratia were isolated from different soils and the guts of invertebrates and characterized by their sensitivity to eight indigenous bacteriophages. They were also classified according to bacteriocin production and sensitivity, BiOLOG plate and API 20E strip profiles and 16S rRNA sequence information. One strain was thus identified as Serratia plymuthica, another as Serratia fonticola. The remaining strains were shown to be closely related to Serratia proteamaculans subsp. quinovora Grimont et al. 1983 after DNA-DNA cross-hybridization demonstrated relatedness greater than 70% with the type strain of this subspecies. From an ecological perspective, our results illustrated the wide variation in sensitivity that closely related Serratia strains have towards various indigenous soil phages and that these phages have broad host ranges within the genus. Furthermore, the phage and bacteriocin interactions within the Serratia strains examined were intricate and did not reflect phylogenetic relationships. These results together imply that complex interactions will occur in soil within the natural community of Serratia strains and their bacteriophages. DNA-DNA cross-hybridization and phenotypic characterization showed that S. proteamaculans subsp. quinovora strains formed a cohesive group at the species level. It is therefore concluded that these strains should be designated as Serratia quinivorans corrig., sp. nov.

Occurrence, Transfer and Mobilization in Epilithic Strains of Acinetobacter of Mercury-resistance Plasmids Capable of Transformation

A 7.8 kb plasmid (pQM17) encoding mercury resistance was isolated from two epilithic strains of Acinetobacter calcoaceticus. The plasmid had a broad host range when mobilized by RP1, transferring into Pseudomonas aeruginosa, P. putida, P. fluorescens, Escherichia coli, Proteus vulgaris and Chromobacterium sp. with frequencies ranging from 5.3 x 10(-9) to 4.6 x 10(-4) per recipient. The plasmid could be transferred into A. calcoaceticus BD413 using intact cells of donor and recipient bacteria (i.e. natural transformation) and there was a broad temperature optimum (14-37 degrees C) for transformation. Transformation was as efficient in liquid matings as on plates but there was no effect of pH in the range 5.6-7.9. Maximum transformation frequencies were obtained after 24 h on agar plates containing 3.5-10 g C 1-1 with donor to recipient ratios ranging from 6 to 415.