Pseudomonas Communities Research Articles

Anti-Virulence Potential of a Chionodracine-Derived Peptide against Multidrug-Resistant Pseudomonas aeruginosa Clinical Isolates from Cystic Fibrosis Patients.

Pseudomonas aeruginosa is an opportunistic pathogen causing several chronic infections resistant to currently available antibiotics. Its pathogenicity is related to the production of different virulence factors such as biofilm and protease secretion. Pseudomonas communities can persist in biofilms that protect bacterial cells from antibiotics. Hence, there is a need for innovative approaches that are able to counteract these virulence factors, which play a pivotal role, especially in chronic infections. In this context, antimicrobial peptides are emerging drugs showing a broad spectrum of antibacterial activity. Here, we tested the anti-virulence activity of a chionodracine-derived peptide (KHS-Cnd) on five P. aeruginosa clinical isolates from cystic fibrosis patients. We demonstrated that KHS-Cnd impaired biofilm development and caused biofilm disaggregation without affecting bacterial viability in nearly all of the tested strains. Ultrastructural morphological analysis showed that the effect of KHS-Cnd on biofilm could be related to a different compactness of the matrix. KHS-Cnd was also able to reduce adhesion to pulmonary cell lines and to impair the invasion of host cells by P. aeruginosa. A cytotoxic effect of KHS-Cnd was observed only at the highest tested concentration. This study highlights the potential of KHS-Cnd as an anti-biofilm and anti-virulence molecule against P. aeruginosa clinical strains.

Erratum to "Positive linkage between bacterial social traits reveals that homogeneous rather than specialized behavioral repertoires prevail in natural Pseudomonas communities".

Erratum to "Positive linkage between bacterial social traits reveals that homogeneous rather than specialized behavioral repertoires prevail in natural Pseudomonas communities".

Positive linkage between bacterial social traits reveals that homogeneous rather than specialised behavioral repertoires prevail in natural Pseudomonas communities.

Bacteria frequently cooperate by sharing secreted metabolites such as enzymes and siderophores. The expression of such 'public good' traits can be interdependent, and studies on laboratory systems have shown that trait linkage affects eco-evolutionary dynamics within bacterial communities. Here, we examine whether linkage among social traits occurs in natural habitats by examining investment levels and correlations between five public goods (biosurfactants, biofilm components, proteases, pyoverdines and toxic compounds) in 315 Pseudomonas isolates from soil and freshwater communities. Our phenotypic assays revealed that (i) social trait expression profiles varied dramatically; (ii) correlations between traits were frequent, exclusively positive and sometimes habitat-specific; and (iii) heterogeneous (specialised) trait repertoires were rarer than homogeneous (unspecialised) repertoires. Our results show that most isolates lie on a continuum between a 'social' type producing multiple public goods, and an 'asocial' type showing low investment into social traits. This segregation could reflect local adaptation to different microhabitats, or emerge from interactions between different social strategies. In the latter case, our findings suggest that the scope for competition among unspecialised isolates exceeds the scope for mutualistic exchange of different public goods between specialised isolates. Overall, our results indicate that complex interdependencies among social traits shape microbial lifestyles in nature.

Pseudomonas-Specific NGS Assay Provides Insight Into Abundance and Dynamics of Pseudomonas Species Including P. aeruginosa in a Cooling Tower.

Pseudomonas species are frequent inhabitants of freshwater environments and colonizers of water supply networks via bioadhesion and biofilm formation. P. aeruginosa is the species most commonly associated with human disease, causing a wide variety of infections with links to its presence in freshwater systems. Though several other Pseudomonas species are of ecological and public health importance, little knowledge exists regarding environmental abundances of these species. In the present study, an Illumina-based next-generation sequencing (NGS) approach using Pseudomonas-specific primers targeting the 16S rRNA gene was evaluated and applied to a set of freshwater samples from different environments including a cooling tower sampled monthly during 2 years. Our approach showed high in situ specificity and accuracy. NGS read counts revealed a precise quantification of P. aeruginosa and a good correlation with the absolute number of Pseudomonas genome copies in a validated genus-specific qPCR assay, demonstrating the ability of the NGS approach to determine both relative and absolute abundances of Pseudomonas species and P. aeruginosa. The characterization of Pseudomonas communities in cooling tower water allowed us to identify 43 phylotypes, with P. aeruginosa being the most abundant. A shift existed within each year from a community dominated by phylotypes belonging to P. fluorescens and P. oleovorans phylogenetic groups to a community where P. aeruginosa was highly abundant. Co-occurrence was observed between P. aeruginosa and other phylotypes of P. aeruginosa group as well as the potentially pathogenic species P. stutzeri, but not with phylotypes of the P. fluorescens group, indicating the need to further investigate the metabolic networks and ecological traits of Pseudomonas species. This study demonstrates the potential of deep sequencing as a valuable tool in environmental diagnostics and surveillance of health-related pathogens in freshwater environments.

Effect of land use and soil organic matter quality on the structure and function of microbial communities in pastoral soils: Implications for disease suppression.

Cropping soils vary in extent of natural suppression of soil-borne plant diseases. However, it is unknown whether similar variation occurs across pastoral agricultural systems. We examined soil microbial community properties known to be associated with disease suppression across 50 pastoral fields varying in management intensity. The composition and abundance of the disease-suppressive community were assessed from both taxonomic and functional perspectives. Pseudomonas bacteria were selected as a general taxonomic indicator of disease suppressive potential, while genes associated with the biosynthesis of a suite of secondary metabolites provided functional markers (GeoChip 5.0 microarray analysis). The composition of both the Pseudomonas communities and disease suppressive functional genes were responsive to land use. Underlying soil properties explained 37% of the variation in Pseudomonas community structure and up to 61% of the variation in the abundance of disease suppressive functional genes. Notably, measures of soil organic matter quality, C:P ratio, and aromaticity of the dissolved organic matter content (carbon recalcitrance), influenced both the taxonomic and functional disease suppressive potential of the pasture soils. Our results suggest that key components of the soil microbial community may be managed on-farm to enhance disease suppression and plant productivity.

Assessment of the Diversity of Pseudomonas spp. and Fusarium spp. in Radix pseudostellariae Rhizosphere under Monoculture by Combining DGGE and Quantitative PCR.

Radix pseudostellariae is a perennial tonic medicinal plant, with high medicinal value. However, consecutive monoculture of this plant in the same field results in serious decrease in both yield and quality. In this study, a 3-year field experiment was performed to identify the inhibitory effect of growth caused by prolonged monoculture of R. pseudostellariae. DGGE analysis was used to explore the shifts in the structure and diversity of soil Fusarium and Pseudomonas communities along a 3-year gradient of monoculture. The results demonstrated that extended monoculture significantly boosted the diversity of Fusarium spp., but declined Pseudomonas spp. diversity. Quantitative PCR analysis showed a significant increase in Fusarium oxysporum, but a decline in Pseudomonas spp. Furthermore, abundance of antagonistic Pseudomonas spp. possessing antagonistic ability toward F. oxysporum significantly decreased in consecutively monocultured soils. Phenolic acid mixture at the same ratio as detected in soil could boost mycelial and sporular growth of pathogenic F. oxysporum while inhibit the growth of antagonistic Pseudomonas sp. CJ313. Moreover, plant bioassays showed that Pseudomonas sp. CJ313 had a good performance that protected R. pseudostellariae from infection by F. oxysporum. In conclusion, this study demonstrated that extended monoculture of R. pseudostellariae could alter the Fusarium and Pseudomonas communities in the plant rhizosphere, leading to relatively low level of antagonistic microorganisms, but with relatively high level of pathogenic microorganisms.

Siderophore cheating and cheating resistance shape competition for iron in soil and freshwater Pseudomonas communities

All social organisms experience dilemmas between cooperators performing group-beneficial actions and cheats selfishly exploiting these actions. Although bacteria have become model organisms to study social dilemmas in laboratory systems, we know little about their relevance in natural communities. Here, we show that social interactions mediated by a single shareable compound necessary for growth (the iron-scavenging pyoverdine) have important consequences for competitive dynamics in soil and pond communities of Pseudomonas bacteria. We find that pyoverdine non- and low-producers co-occur in many natural communities. While non-producers have genes coding for multiple pyoverdine receptors and are able to exploit compatible heterologous pyoverdines from other community members, producers differ in the pyoverdine types they secrete, offering protection against exploitation from non-producers with incompatible receptors. Our findings indicate that there is both selection for cheating and cheating resistance, which could drive antagonistic co-evolution and diversification in natural bacterial communities.

Long- term grapevine cultivation and agro-environment affect rhizosphere microbiome rather than plant age

Single plant replacement is one of the most commonly adopted practices to overcome vineyard crop decline associated with grapevine trunk diseases. A study was undertaken in long-term cultivated vineyards of an important wine producing district in northern Italy to investigate whether rhizosphere microbiome differed between adult and young replaced plants.Fungal and bacterial communities in adult and young replaced plants were investigated in ten vineyards across four geographic areas within the district. General and specific fungal and bacterial primers were used for PCR-DGGE and qPCR analysis. Moreover, quali-quantitative evaluation of Cylindrocarpon-like fungi, agents of foot rot belonging to the disease complex involved in vineyard decline, was performed with specific primers using nested PCR.Fungal and bacterial communities did not differ between adult and young plants, conversely they differed in geographic areas. While Pseudomonas and Bacillus communities varied mostly according to geographic origin, Ascomycota (microfungi) and Basidiomycota (white-rot fungi) also varied between the vineyards within each area. Furthermore, Cylindrocarpon-like species showed an ubiquitous occurrence, regardless plant age.Results show that long-term plant growth legacy overcomes plant age in shaping rhizosphere microbiome. Although composition of rhizosphere microbiome does not seem to represent a bio-indicator of vineyard decline according to findings of this study, the site-dependent variation of microbiome indicates that part of the complex phenomenon of crop decline may be due to a variable interaction between a series of soil biotic components and physiological state of grapevines.

Probiotic Pseudomonas communities enhance plant growth and nutrient assimilation via diversity-mediated ecosystem functioning

Plant-associated microbes play an important role in plant growth and development. While the introduction of beneficial microbes into the soil could improve plant production in low-input agricultural systems, real-world applications are still held back by poor survival and activity of the probiotic microbes. In this study, we used a biodiversity-ecosystem functioning (BEF) framework to specifically test how Pseudomonas community richness shapes the bacterial inoculant survival and functioning in terms of plant growth. To this end, we manipulated the richness of a probiotic Pseudomonas spp. bacterial community inoculant (1, 2, 4 or 8 strains per community) and compared diversity and strain identity effects on plant biomass production and nutrient assimilation in vivo with tomato. We found that increasing the richness of the bacterial inoculant enhanced the survival and abundance of Pseudomonas communities leading to higher accumulation of plant biomass and more efficient assimilation of nutrients into the plant tissue. Diversity effects were clearly stronger than the Pseudomonas strain identity effects and diversity-mediated plant growth promotion could be linked with increased production of plant hormones, siderophores and solubilization of phosphorus in vitro. Together these results suggest that multi-strain microbial inoculants can promote plant growth more reliably and effectively compared to single-strain inoculants.

Impacts of endophyte infection of ryegrass on rhizosphere metabolome and microbial community

The use of grasses such as ryegrass and fescues infected with endophytic fungi of the Epichloë genus is widespread in New Zealand’s pastoral systems. Each endophyte–cultivar combination represents a distinctive genome–genome association, resulting in unique biological outcomes. The wider influence of these interactions on rhizosphere microbiology are not well characterised. This is important, because there may be opportunities or risks associated with selective disruption of the rhizosphere microbiota. We explored the interaction of two commercially used endophyte fungi, E. festucae var. lolii strains AR1 and AR37, within a genetically uniform breeding line of perennial ryegrass (Lolium perenne cv. Samson 11104) on the rhizosphere metabolome and the composition of the fungal, bacterial, and Pseudomonas communities. There were strong differences in the rhizosphere metabolomes between infested and non-infested ryegrass strains (P = 0.06). These were attributed to shifts in various n-alkane hydrocarbon compounds. The endophyte-associated alteration in rhizosphere metabolome was linked to changes in the total bacterial (P < 0.01) and fungal (P < 0.05) rhizosphere communities. Furthermore, there was varying levels of support for endophyte-specific (AR1 v. AR37) impacts on the bacterial and fungal communities. Pseudomonas bacterial communities were not influenced by endophyte infection of ryegrass (P = 0.834).

Biodiversity and species identity shape the antifungal activity of bacterial communities.

Soils host diverse communities of interacting microbes and the nature of interspecific interactions is increasingly recognized to affect ecosystem-level processes. Antagonistic interactions between bacteria and fungi are of particular relevance for soil functioning. A number of soil bacteria produce secondary metabolites that inhibit eukaryotic growth. Antibiosis may be stimulated in the presence of competing bacteria, and we tested if biodiversity within bacterial communities affects their antagonistic activity against fungi and fungal-like species. We set up Pseudomonas communities of increasing diversity and measured the production of the broad spectrum antifungal compound 2,4-DAPG and their antagonistic activity against different eukaryotes. Diversity increased DAPG concentration and antifungal activity, an effect due to a combination of identity and interactions between species. Our results indicate that investment of pseudomonads into broad spectrum anti-eukaryotic traits is determined by both community composition and diversity and this provides new avenues to understand interactions between bacterial and fungal communities.

The freshwater sponge Ephydatia fluviatilis harbours diverse Pseudomonas species (Gammaproteobacteria, Pseudomonadales) with broad-spectrum antimicrobial activity.

Bacteria are believed to play an important role in the fitness and biochemistry of sponges (Porifera). Pseudomonas species (Gammaproteobacteria, Pseudomonadales) are capable of colonizing a broad range of eukaryotic hosts, but knowledge of their diversity and function in freshwater invertebrates is rudimentary. We assessed the diversity, structure and antimicrobial activities of Pseudomonas spp. in the freshwater sponge Ephydatia fluviatilis. Polymerase Chain Reaction – Denaturing Gradient Gel Electrophoresis (PCR-DGGE) fingerprints of the global regulator gene gacA revealed distinct structures between sponge-associated and free-living Pseudomonas communities, unveiling previously unsuspected diversity of these assemblages in freshwater. Community structures varied across E. fluviatilis specimens, yet specific gacA phylotypes could be detected by PCR-DGGE in almost all sponge individuals sampled over two consecutive years. By means of whole-genome fingerprinting, 39 distinct genotypes were found within 90 fluorescent Pseudomonas isolates retrieved from E. fluviatilis. High frequency of in vitro antibacterial (49%), antiprotozoan (35%) and anti-oomycetal (32%) activities was found among these isolates, contrasting less-pronounced basidiomycetal (17%) and ascomycetal (8%) antagonism. Culture extracts of highly predation-resistant isolates rapidly caused complete immobility or lysis of cells of the protozoan Colpoda steinii. Isolates tentatively identified as P. jessenii, P. protegens and P. oryzihabitans showed conspicuous inhibitory traits and correspondence with dominant sponge-associated phylotypes registered by cultivation-independent analysis. Our findings suggest that E. fluviatilis hosts both transient and persistent Pseudomonas symbionts displaying antimicrobial activities of potential ecological and biotechnological value.

Response of soil microbial communities to contrasted histories of phosphorus fertilisation in pastures

Several microbial taxa that potentially affect soil phosphorus (P) biogeochemical cycling and plant nutrition have been identified, in particular some members of the Actinobacteria, Pseudomonas, and arbuscular mycorrhizal fungi (AMF) have been found to solubilise P and enhance P uptake. This study investigated the diversity of selected key taxa within a pastoral agricultural system, to address the hypothesis that if these taxa have an intrinsic association to soil P, long-term P-manipulation should present a selective pressure. We defined this selective pressure as a shift in community assemblage, based on either PCR-DGGE banding patterns or taxon-specific quantitative polymerase chain reaction (qPCR) (mycorrhizae). The work was conducted on a long-term fertiliser field trial site where soil P status had been altered for ∼40 years, with total P ranging from 464 to 1102mgkg−1 soil. The structure of both Actinobacteria and Pseudomonas communities was found to differ according to fertiliser treatment (P<0.05). However, only the Actinobacteria were associated with the underlying P fertility gradient (soil C:P ratio; ρ=0.481; P=0.019); no significant links between the Pseudomonas community and soil physicochemical properties were found. As the structure of the general bacterial or α-Proteobacterial communities did not vary between treatments (P>0.05), the hypothesis that Actinobacteria communities are intrinsically linked to soil P cycling is supported. Similarly, mycorrhizal community structure was strongly influenced by soil P status (P=0.002), with particularly strong differences between the control treatments (no P fertiliser) and fertilised plots, meaning the addition of fertiliser P to soil per se was more important than quantity. P-fertilizer application increased the diversity of AMF. The AMF community of the control plot was dominated by Glomus intraradices and Glomus claroideum; these taxa may have had the highest fitness under the low P conditions. The total fungal community structure, assessed by denaturing gradient gel electrophoresis (DGGE), also varied over the fertiliser trial (P=0.001), however the variation was not correlated with soil P status (biota and/or environment matching; BIO-ENV test). Our results expand our knowledge on the microbiology supporting soil P cycling in pasture soils. Furthermore, we demonstrate that communities of Actinobacteria and AMF, proposed to be involved in supplying P to pasture plants, are linked to the underlying soil P status.

PseudomonasDiversity in Crude-Oil-Contaminated Intertidal Sand Samples Obtained after thePrestigeOil Spill

The Galicia seashore, in northwestern Spain, was one of the shorelines affected by the Prestige oil spill in November 2002. The diversity of autochthonous Pseudomonas populations present at two beaches (Carnota municipality) was analyzed using culture-independent and culture-dependent methods. The first analysis involved the screening of an rpoD gene library. The second involved the isolation of 94 Pseudomonas strains that were able to grow on selective media by direct plating or after serial enrichments on several carbon sources: biphenyl, gentisate, hexadecane, methylnaphthalene, naphthalene, phenanthrene, salicylate, xylene, and succinate. Eight denitrifying Pseudomonas strains were also isolated by their ability to grow anaerobically with nitrate. The calculated coverage index for Pseudomonas species was 89% when clones and isolates were considered together, and there were 29 phylospecies detected. The most abundant were members of the species P. stutzeri, P. putida, P. anguilliseptica, and P. oleovorans. Thirty-one isolates could not be identified at the species level and were considered representatives of 16 putative novel Pseudomonas species. One isolate was considered representative of a novel P. stutzeri genomovar. Concordant results were obtained when the diversities of the cloned DNA library and the cultured strains were compared. The clone library obtained by the rpoD PCR method was a useful tool for evaluating Pseudomonas communities and also for microdiversity studies of Pseudomonas populations.

Universal and species‐specific bacterial ‘fungiphiles’ in the mycospheres of different basidiomycetous fungi

In previous work, several bacterial groups that show a response to fruiting bodies (the mycosphere) of the ectomycorrhizal fungus Laccaria proxima were identified. We here extend this work to a broader range of fungal fruiting bodies sampled at two occasions. PCR-DGGE analyses showed clear effects of the mycosphere of diverse fungi on the total bacterial and Pseudomonas communities in comparison with those in the corresponding bulk soil. The diversities of the Pseudomonas communities increased dramatically in most of the mycospheres tested, which contrasted with a decrease of the diversity of the total bacterial communities in these habitats. The data also indicated the existence of universal (i.e. Pseudomonas poae, P. lini, P. umsongensis, P. corrugata, P. antarctica and Rahnella aquatilis) as well as specific (i.e. P. viridiflava and candidatus Xiphinematobacter americani) fungiphiles, defined as bacteria adapted to the mycospheres of, respectively, three or more or just one fungal species. The selection of such fungiphiles was shown to be strongly related to their capacities to use particular carbonaceous compounds, as evidenced using principal components analyses of BIOLOG-based substrate utilization tests. The differentiating compounds, i.e. L-arabinose, L-leucine, m-inositol, m-arabitol, D-mannitol and D-trehalose, were tentatively linked to compounds known to occur in mycosphere exudates.

Request from the European Commission related to the safeguard clause invoked by Hungary on maize MON810 according to Article 23 of Directive 2001/18/EC - Scientific opinion of the Panel on Genetically Modified Organisms.

Request from the European Commission related to the safeguard clause invoked by Hungary on maize MON810 according to Article 23 of Directive 2001/18/EC - Scientific opinion of the Panel on Genetically Modified Organisms.

Effects of plant genotype and growth stage on the structure of bacterial communities associated with potato (Solanum tuberosum L.)

The effects of genotype, plant growth and experimental factors (soil and year) on potato-associated bacterial communities were studied. Cultivars Achirana Inta, Désirée, Merkur and transgenic Désirée line DL12 (containing T4 lysozyme gene) were assessed in two field experiments. Cross-comparisons between both experiments were made using Désirée plants. Culture-dependent and -independent approaches were used to demonstrate effects on total bacterial, actinobacterial and Pseudomonas communities in bulk and rhizosphere soils and endospheres. PCR-denaturing gradient gel electrophoresis fingerprints prepared with group-specific primers were analyzed using multivariate analyses and revealed that bacterial communities in Achirana Inta plants differed most from those of Désirée and Merkur. No significant effects were found between Désirée and DL12 lines. Plant growth stage strongly affected different plant-associated communities in both experiments. To investigate the effect of plant-associated communities on plant health, 800 isolates from rhizospheres and endospheres at the flowering stage were tested for suppression of Ralstonia solanacearum biovar 2 and/or Rhizoctonia solani AG3. A group of isolates closely resembling Lysobacter sp. dominated in young plants. Its prevalence was affected by plant growth stage and experiment rather than by plant genotype. It was concluded that plant growth stage overwhelmed any effect of plant genotype on the bacterial communities associated with potato.

Characterization ofBacillusandPseudomonasstrains with suppressive traits isolated from tomato hydroponic-slow filtration unit

Bacillus and Pseudomonas spp. are known to be involved in plant pathogenic fungi elimination during the slow filtration process used in tomato soilless cultures. We isolated 6-8 strains of both Bacillus and Pseudomonas from the top, middle, and bottom sections of filters and identified them after 16S rDNA sequencing. Four Pseudomonas strains were identified as Pseudomonas fulva, 5 as Pseudomonas plecoglossicida, and 12 as Pseudomonas putida. The use of specific oligonucleotide polymerase chain reaction primer sets designed from gyrB gene sequences additionally permitted the identification of 17 Bacillus cereus and 3 Bacillus thuringiensis strains. Ribotyping with EcoRI pointed out an important polymorphism within Bacillus and Pseudomonas strains. Molecular characterization did not reveal a correlation between the location of isolates within the filter (top, middle, or bottom) and bacterial identification or riboclusters. Functional aspects assessed by community-level physiological profiling showed marked phenotypic differences between Pseudomonas communities isolated from the top and bottom filter layers; differences were lower between Bacillus communities of different layers and far less noticeable between mixed communities of Bacillus and Pseudomonas. These strains were tested for several suppressive activities. Conversely to most Bacillus, the majority of Pseudomonas strains were auxin producers and promoted the growth of tomato plantlet roots. On the other hand, only Bacillus strains displayed antagonistic activities by inhibiting the growth of pathogenic fungi frequently detected in soilless cultures. Siderophores were produced by nearly all bacteria, but at higher amounts by Pseudomonas than Bacillus strains. The biocontrol agent potentiality of certain strains to optimize the slow filtration process and to promote the suppressive potential of nutrient solution is discussed.

Effect of above‐ground plant species on soil microbial community structure and its impact on suppression of Rhizoctonia solani AG3

The extent of soil microbial diversity is seen to be critical to the maintenance of soil health and quality. Different agricultural practices are able to affect soil microbial diversity and thus the level of suppressiveness of plant diseases. In a 4-year field experiment, we investigated the microbial diversity of soil under different agricultural regimes. We studied permanent grassland, grassland turned into arable land, long-term arable land and arable land turned into grassland. The diversity of microbial communities was described by using cultivation-based and cultivation-independent methods. Both types of methods revealed differences in the diversities of soil microbial communities between different treatments. The treatments with higher above-ground biodiversity generally maintained higher levels of microbial diversity. Moreover, a positive correlation between suppression of Rhizoctonia solani AG3 and microbial diversity was observed. Permanent (species-rich) grassland and grassland turned into maize stimulated higher microbial diversities and higher levels of suppressiveness of R. solani AG3 compared with the long-term arable land. Effects of agricultural practices on Bacillus and Pseudomonas communities were also observed and clear correlations between the levels of suppressiveness and the diversities of these bacterial groups were found. This study highlighted the importance of agricultural management regime for soil microbial community structure and diversity as well as the level of soil suppressiveness.

Influence of soil sample sizes on the assessment of bacterial community structure

When assessing bacterial community structure in soil it is important to establish a satisfactory procedure for sampling. The influence of sample sizes of a forest soil on the assessment of bacterial community structure was investigated. Four sample sizes (0.01, 0.1, 1.0, and 10.0 g) were evaluated. Time of colony appearance on a nutrient-limited soil extract agar was used to characterise the culturable heterotrophic and Pseudomonas communities. Genetic community structure was assessed with denaturing gradient gel electrophoresis (DGGE) of 16S rDNA using a bacterial primer set. The largest variation in the heterotrophic community structure for bacteria was seen when comparing the 0.01 g replicates. Variation was also seen for the 0.1 and 1.0 g replicates. However, there was no significant difference between the 10.0 g replicates. The 0.01 and 0.1 g replicates showed variation in genetic community structure within the sample sizes, whereas variation between the replicates within the larger sample sizes (1.0, 10.0 g) was negligible. Variation in the heterotrophic community structure for Pseudomonas was seen between replicates of all sample sizes. Hence, the size of soil samples influenced the bacterial community structure observed for bacteria, whereas chance seemed to play an important role when looking at a more narrow community structure.