Analysis of a Pool of Small Plasmids from Soil Heterotrophic Cultivable Bacterial Communities

Soil Sampling and Processing

Bacteria analyzed in this work (Table 1) were isolated from six soil samples collected from the top and the bottom of a 13-years experimental field located in the coastal hills of Marche (Agugliano, Ancona, Central Italy). The six soil samples were referred to as 2.2 A, 2.2 B, 2.2 C and 4.1 A, 4.1 B and 4.1 C (from the top and the bottom of the experimental field, respectively). The Agugliano soil is a Calcaric Gleyic Cambisol with 20% slope [9], which in the first 30 cm has an Ap horizon (that is the homogeneous layer due to plowing) and a clay-silt texture [10]. The soil is managed under a Triticum durum (in winter) and Zea mays (in summer) rotation. Six soil samples were collected on 11 June 2007, during the maize rotation, in no-tillage (NT) system (sod seeding with chemical desiccation and chopping) and unfertilized (UF) soil (0 Kg N ha^-1) at 0-20 cm depth. Each soil sample consisted of five soil cores taken inside two NT-UF blocks (top and bottom) of experimental field free from roots and then pooled together. Soil samples were sieved immediately at 2-mm mesh size, kept at 4°C and processed for further analysis within 24 h from sampling.

Culturing of Fast- and Slow-growing Culturable Bacteria

About 1-g (wet weight) of each soil sample was suspended in 10 ml of sterile phosphate-buffered saline (PBS, pH 7.3), homogenized at low speed 3 (Ultra-Turrax Thyristor Regle 50, Janke & Kunkel IKA-Labortechnik) and vortexed for 30 seconds. Then, each sample was transferred into a sterile 100-ml Erlenmeyer flask containing 10 g of glass beads (average diameter, 2 mm previously autoclaved for 20 min at 121°C) and shaken for 1 h at 120 rpm and 28°C to disperse bacteria. The flasks and glass beads were autoclaved for 20 min at 121°C before use. The resulting soil suspension was removed and transferred to a sterile 15-ml Falcon tube. Serial dilutions of this suspension were performed with sterile saline solution (9 g l^-1NaCl) from 10^-1up to 10^-7. Then, 100 μl aliquots of serially diluted soil suspensions were plated in triplicate on 0.1 tryptic soy broth (TSB, Difco) containing 15 g l^-1 agar (0.1 TSA) and 100 ìg ml^-1 cycloheximide (Sigma) to inhibit fungal growth. Plates were incubated at 28 °C for 6 days. Total culturable bacteria were enumerated on the basis of the r/K strategy concept [11] at day 1, 2 and 6; in this way, three counts per plate were performed, corresponding to three classes (1, 2, and 3) with different growth rate. Bacteria producing visible colonies at days 1 and 2 (classes 1 and 2) were defined as “fast growers” (copiotrophs or r-strategists), while bacteria that produced colonies later (class 3) were defined as “slow growers” (oligotrophs or K-strategists). The number of bacteria in each class was expressed as a percentage of the total count and gave insight into the distribution of r- and K-strategists in each sample. Characteristics of r-strategists include fast growth in response to medium enrichment, while K-strategists are characterized by slow growth in response to enrichment.

After six days of growth at 28°C, a set of 1,200 colonies (200 per each of the six soil samples) were randomly chosen from 0.1 TSA plates and isolated on the same medium for the further characterization.

Eco-physiological Index

To express the distribution of the fast- versus slow-growing bacteria (r-versusK- strategists) in soil samples, the Eco-Physiological (EP) index [12], was calculated using three classes (i.e. colonies grown after 1, 2, and 6 days) [11]. The EP index of each soil tested was calculated using the equation: H’= -∑(P_i x log₁₀ P_i), where P_i represents the CFU at each day (1, 2 and 6 days of incubation) as a proportion of the total CFU in that sample after 6 days incubation i.e. the proportion of colonies appearing on counting day i (i = 1, 2, 6) with EP_min = 0. Higher values of EP index imply a more even distribution of proportions of bacteria developing on different days (i.e., different classes of bacteria).

Statistics

Bacterial population data (CFU g^-1 of soil) were log transformed and subsequently analysed by using t-test (Graph Pad Prism version 5 software). Percentage data of r/K strategists and EP index value were logit-transformed, Logit (p) = log [p/(1-1-p)] for the proportion p, and compared using t-test (Graph Pad Prism version 5 software).

Agarose Gel Electrophoresis

Agarose gel electrophoresis in TAE buffer (0.04 M Tris-Acetate, 0.01 M EDTA) containing 0.5 μg/ml (w/v) of ethidium bromide [13] was used to check the presence of plasmids (0.8% w/v), and to analyze amplicons obtained either from PCR amplification of 16S rRNA genes (0.8% w/v) or RAPD fingerprinting (2.0 % w/v).

Analysis of Plasmids Content

Analytical amounts of plasmid DNA were obtained from 1.5 ml bacterial cultures using the commercial Kit Plasmid Miniprep (Qiagen) set up for Gram-negative bacteria with the use of a robotic workstation (QiaCube, Qiagen).

PCR Amplification and Sequencing of 16S rRNA Genes

PCR amplification of 16S rRNA genes was carried out according to Papaleo et al. [14] using a MJ Research PTC 100 Peltier Thermal Cycler (CELBIO). Amplicons were excised from agarose gel and purified using the “QIAquick” gel extraction kit (QiAgen). Direct sequencing was performed on both DNA strands using the chemical dye terminator [15].

RAPD Analysis

Random amplification of DNA fragments was carried out using primer 1253 (5’ GTTTCCGCCC 3’) and the amplification conditions described elsewhere [16].

Homologs Retrieval and Phylogenetic Analysis

Probing of the DNA databases was performed with the BLAST program [17], using default parameters. The ClustalW program [18] was used to align the 16S rRNA gene sequences obtained with the most similar ones retrieved from databases. Each alignment was analyzed using the neighbor-joining method [19] according to the model of Kimura 2-parameter distances [20]. Phylogenetic trees were constructed using the MEGA4 software [21]. The robustness of the inferred trees was evaluated by 1000 bootstrap resamplings.

Characterization of Bacterial Communities Isolated from Soil Samples

Six soil samples were collected from two boxes of UF-NT soil, i.e. from the top (2.2 A, 2.2 B, and 2.2 C samples) and from the bottom (4.1 A, 4.1 B, and 4.1 C samples) of the hillside. The total microbial population density ranged from Log₁₀ 6.01 ± 0.05 (bottom) to Log₁₀ 6.61 ± 0.17 (top) cfu g^-1 of soil (Table 2), and no significant difference (P>0.05) was observed between the soil samplings collected from the top and the bottom of the experimental field. The structure of the bacterial soil community at each sub-sampling was investigated using the concept of r/K strategy [11] and reported in Table 3. Results indicated that bacterial colonies visible after one day of incubation (r/K class 1) were more abundant in the bottom soil compared to those in the top soil (P<0.05), whereas no differences (P>0.05) were observed between the bottom and the top samplings colonies visible after two days of incubation (r/K classes 2 and 3) (Table 3). Significant differences in EPI-index between the top and the bottom soil were also found (P<0.05) (Table 3), suggesting changes in community structure of cultivable bacterial communities in the two blocks of experimental field.

Analysis of Plasmid Content

Two hundreds bacterial isolates from each of the six samples (2.2 A, 2.2 B, 2.2 C, 4.1 A, 4.1 B, and 4.1. C) were randomly selected for further characterization. The presence of plasmids was checked as described in Materials and Methods on each of the 1,200 bacterial isolates randomly selected and re-grown on 0.1 TSA medium (Tables 1 and 4). Data obtained are shown in Fig. (2) and revealed that only 27 out of the 1,200 bacterial isolates harbored plasmid molecules. Most of the strains exhibited only a single plasmid molecule, while in a few cases (i.e. isolates 308bis and 363) multiple plasmids were found in the same cell. Furthermore, some isolates showed plasmids with the same electrophoretic mobility. The size of plasmid molecules ranged between about 2 Kb and 40 Kb, as determined by comparing their eletrophoretic mobility with that of reference plasmids. However, we cannot a priori exclude the possibility that the genome of some of the bacterial isolates analyzed might contain large and/or low copy number plasmids, which might have not been revealed by the extraction methodology used in this work.

RAPD Fingerprinting

In order to type the 27 bacterial isolates harboring plasmids, a RAPD [16] analysis using the primer 1253 was carried out. The comparative analysis of RAPD profiles obtained allowed the bacterial strains to be clustered in groups embedding bacterial isolates exhibiting the very same amplification profile (hereinafter haplotype). Bacterial isolates with the same haplotype were considered as the same strain. Data obtained are reported in Fig. (2), which shows that the 27 isolates can be split into 15 RAPD groups. Indeed, some isolates exhibited the same RAPD profile, suggesting that they might correspond to the same bacterial strain. In most cases, isolates exhibiting the same RAPD profile share a plasmid with the same electrophoretic mobility (i.e. the same plasmid if we assume that plasmids with the same electrophoretic mobility correspond to the same molecule) (see, for instance, isolates 230, 251, 253, 327 and 398 – RAPD haplotype 3). Just in one case, isolates sharing the same RAPD profile (i.e. cells of the same strain) harbored different plasmids (isolates 457, 500, 506 - RAPD haplotype 13), suggesting that the same strain may host different plasmids.

Phylogenetic Affiliation of Bacterial Isolates Harboring Plasmid Molecules

To affiliate each bacterial isolate to a given taxon, the nucleotide sequence of the 16S rRNA gene from at least one isolate per each RAPD group was determined. The 16S rRNA genes were PCR-amplified and sequenced from 25 isolates and their analysis revealed that:

i) The 15 RAPD haplotypes were representative of seven bacterial genera, two Gram positive (Staphylococcus and Paenibacillus) and five Gram negative (Acinetobacter, Enterobacter, Pantoea, Stenotrophomonas, and Klebsiella, all belonging to (-proteobacteria), with Acinetobacter and Paenibacillus representing half of the bacteria-harboring plasmids community (Fig. 2 and Table 4).

ii) The 16S rRNA gene sequences from three Enterobacter isolates (457, 500, 506) were identical in agreement with the finding that they also share the same RAPD profile. The fourth Enterobacter isolate (363), exhibiting a different RAPD profile (Fig. 2), also possesses a 16S rRNA gene sequence differing in one position in respect to the other three ones.

iii) The two Staphylococcus strains (706 and 708 exhibiting different RAPD haplotypes) shared the same 16S rRNA gene sequence, suggesting that they belong to the same species.

iv) The Acinetobacter sequences were placed in three distant branches of the trees, suggesting that they very likely belong to (at least) three different species, a finding that is in agreement with their very different RAPD profile.

Analysis of the Composition of the Aerobic Heterotrophic Cultivable Bacterial Communities Lacking Small Plasmids

In order to get some information also on the composition of the heterotrophic cultivable bacterial community, which did not exhibit plasmids under the experimental conditions used in this work and to compare it with the taxonomical position of bacteria harboring small plasmids, the 16S rRNA genes were amplified and sequenced as described in Materials and Methods from a panel of 79 randomly chosen bacterial isolates (Table 1). The analysis of the 79 nucleotide sequences obtained revealed that they were affiliated to 14 bacterial genera, with Bacillus (21 isolates) and Stenotrophomonas (15 isolates) being the most represented ones. Half of the isolates belong to Gram- bacteria represented only by members of the α- and γ-proteobacteria; the other isolates belong mainly to Bacillus and Paenibacillus, even though representatives of different genera (Agromyces, Arthrobacter, Microbacterium, Rhodococcus, and Streptomyces) of high GC Gram+ bacteria were disclosed.

Data reported in Table 4 revealed that bacteria belonging to Stenotrophomonas, Acinetobacter, Enterobacter and Paenibacillus included both isolates harboring or lacking plasmid molecules. To check the existence of a possible correlation between the presence of plasmids and the phylogenetic position of bacterial isolates, a phylogenetic tree for each of these four genera was constructed (Fig. 3), whose analysis revealed that some isolates embedded in the same genus very likely belong to different species, since the sequences joined different clusters of a phylogenetic tree. This is particularly true for Enterobacter and Acinetobacter isolates, whereas Stenotrophomonas and Paenibacillus exhibited a more homogeneous distribution within the respective tree.