Bacterial diversity promotes community stability and functional resilience after perturbation.

The relationships between bacterial community diversity and stability were investigated by perturbing soils, with naturally differing levels of diversity, to equivalent toxicity using copper sulfate and benzene. Benzene amendment led to large decreases in total bacterial numbers and biomass in both soils. Benzene amendment of an organo-mineral/improved pasture soil altered total soil bacterial community structure but, unlike amendment of the mineral/arable soil, maintained genetic diversity, based on polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis targeting DNA and RNA, until week 9 of the perturbation experiment. Assuming equivalent toxicity, the genetic diversity of the naturally more diverse soil was more resistant to benzene perturbation than the less diverse soil. The broad scale function (mineralization of 14C-labelled wheat shoot) of both benzene- and copper-treated soil communities was unaffected. However, narrow niche function (mineralization of 14C-labelled 2,4-dichlorophenol) was impaired for both benzene-polluted soils. The organo-mineral soil recovered this function by the end of the experiment but the mineral soil did not, suggesting greater resilience in the more diverse soil. Despite a large reduction in bacterial numbers and biomass in the copper-treated soils, only small differences in bacterial community diversity were observed by week 9 in the copper-polluted soils. The overall community structure was little altered and functionality, measured by mineralization rates, remained unchanged. This suggested a non-selective pressure and a degree of genetic and functional resistance to copper perturbation, despite a significant reduction in bacterial numbers and biomass. However, initial shifts in physiological profiles of both copper-polluted soils were observed but rapidly returned to those of the controls. This apparent functional recovery, accompanied by an increase in culturability, possibly reflects adaptation by the surviving communities to perturbation. The findings indicate that, although soil communities may be robust, relationships between diversity and stability need to be considered in developing a predictive understanding of response to environmental perturbations.

The relationship between microbial community structure and functional stability, tested experimentally in an upland pasture soil.

Soil collected from an upland pasture was manipulated experimentally in ways shown previously to alter microbial community structure. One set of soil was subjected to chloroform fumigation for 0, 0.5, 2, or 24 h and the other was sterilised by gamma-irradiation and inoculated with a 10(-2), 10(-4), 10(-6), or 10(-8) dilution of a soil suspension prepared from unsterilized soil. Following incubation for 8 months, to allow for the stabilization of microbial biomass and activity, the resulting microbial community structure (determined by PCR-DGGE of bacterial specific amplification products of total soil DNA) was assessed. In addition, the functional stability (defined here as the resistance and resilience of short-term decomposition of plant residues to a transient heat or a persistent copper perturbation) was determined. Changes in the active bacterial population following perturbation (determined by RT-PCR-DGGE of total soil RNA) were also monitored. The manipulations resulted in distinct shifts in microbial community structure as shown by PCR-DGGE profiles, but no significant decreases in the number of bands. These shifts in microbial community structure were associated with a reduction in functional stability. The clear correlation between altered microbial community structure and functional stability observed in this upland pasture soil was not evident when the same protocols were applied to soils in other studies. RT-PCR-DGGE profiles only detected a shift in the active bacterial population following heat, but not copper, perturbation. We conclude that the functional stability of decomposition is related to specific components of the microbial community.

Spatial structure in soil chemical and microbiological properties in an upland grassland.

We characterised the spatial structure of soil microbial communities in an unimproved grazed upland grassland in the Scottish Borders. A range of soil chemical parameters, cultivable microbes, protozoa, nematodes, phospholipid fatty acid (PLFA) profiles, community-level physiological profiles (CLPP), intra-radical arbuscular mycorrhizal community structure, and eubacterial, actinomycete, pseudomonad and ammonia-oxidiser 16S rRNA gene profiles, assessed by denaturing gradient gel electrophoresis (DGGE) were quantified. The botanical composition of the vegetation associated with each soil sample was also determined. Geostatistical analysis of the data revealed a gamut of spatial dependency with diverse semivariograms being apparent, ranging from pure nugget, linear and non-linear forms. Spatial autocorrelation generally accounted for 40-60% of the total variance of those properties where such autocorrelation was apparent, but accounted for 97% in the case of nitrate-N. Geostatistical ranges extending from approximately 0.6-6 m were detected, dispersed throughout both chemical and biological properties. CLPP data tended to be associated with ranges greater than 4.5 m. There was no relationship between physical distance in the field and genetic similarity based on DGGE profiles. However, analysis of samples taken as close as 1 cm apart within a subset of cores suggested some spatial dependency in community DNA-DGGE parameters below an 8 cm scale. Spatial correlation between the properties was generally weak, with some exceptions such as between microbial biomass C and total N and C. There was evidence for scale-dependence in the relationships between properties. PLFA and CLPP profiling showed some association with vegetation composition, but DGGE profiling did not. There was considerably stronger association between notional sheep urine patches, denoted by soil nutrient status, and many of the properties. These data demonstrate extreme spatial variation in community-level microbiological properties in upland grasslands, and that despite considerable numeric ranges in the majority of properties, overarching controlling factors were not apparent.

Molecular detection and beta-glucuronidase expression of gus-marked Bacillus subtilis L-form bacteria in developing Chinese cabbage seedlings.

AIM: To detect L-form bacteria in developing Chinese cabbage seedlings. METHODS AND RESULTS: Stable Bacillus subtilis L-forms were genetically modified to express the gus gene (encoding beta-glucuronidase). Germinated seeds of Chinese cabbage were soaked in mannitol based suspensions of the L-form bacteria or with mannitol alone and after washing were grown in aseptic conditions on plant growth medium. Histochemical staining of beta-glucuronidase activity (X-gluc) and Polymerase Chain Reaction (PCR) detection of the gus gene were achieved in the L-form associated seedlings. beta-Glucuronidase was localized in discrete spots, mainly in the roots with staining, and was also observed in the cotyledons and base of stems. Correlation was observed between PCR detection of the gus gene and histochemical staining with detection in similar tissues. Stable L-form bacteria were non-culturable after their association with plant material. CONCLUSIONS: The gus reporter gene system with its associated histological staining for enzyme activity was used successfully for detecting B. subtilis L-form bacteria in plant material. SIGNIFICANCE AND IMPACT OF THE STUDY: These molecular marked L-forms should provide a specific and sensitive technique for detecting L-form bacteria in planta and offer a method for further understanding the L-form/plant association.

Novel cyanobacterial biosensor for detection of herbicides.

The aim of this work was to generate a cyanobacterial biosensor that could be used to detect herbicides and other environmental pollutants. A representative freshwater cyanobacterium, Synechocystis sp. strain PCC6803, was chromosomally marked with the luciferase gene luc (from the firefly Photinus pyralis) to create a novel bioluminescent cyanobacterial strain. Successful expression of the luc gene during growth of Synechocystis sp. strain PCC6803 cultures was characterized by measuring optical density and bioluminescence. Bioluminescence was optimized with regard to uptake of the luciferase substrate, luciferin, and the physiology of the cyanobacterium. Bioassays demonstrated that a novel luminescent cyanobacterial biosensor has been developed which responded to a range of compounds including different herbicide types and other toxins. This biosensor is expected to provide new opportunities for the rapid screening of environmental samples or for the investigation of potential environmental damage.

On-line microbial biosensing and fingerprinting of water pollutants.

The potential for biosensors to contribute to on-line toxicity testing for monitoring of water quality is currently constrained both by the relevance of the biosensors available and the technology for biosensor delivery. This paper reports the use of novel slow release biosensor delivery for on-line monitoring instrumentation, with environmentally relevant bacteria for both simple toxicity testing and more complex toxicity fingerprinting of industrial effluents. The on-line toxicity test, using bioluminescence-based biosensors, proved to be as sensitive and reliable as the corresponding batch test, with comparable contaminant EC(50) values from both methods. Toxicity fingerprinting through the investigation of the kinetics (dose-response) and the dynamics (response with time) of the biosensor test response proved to be diagnostic of both effluent type and composition. Furthermore, the slow release of biosensors immobilised in a polyvinyl alcohol (PVA) matrix greatly improved biosensor delivery, did not affect the sensitivity of toxicity testing, and demonstrated great potential for inclusion in on-line monitoring instrumentation.

Toxicity of the bacterial luciferase substrate, n-decyl aldehyde, to Saccharomyces cerevisiae and Caenorhabditis elegans.

This study determined that the bacterial luciferase fusion gene (luxAB) was not a suitable in vivo gene reporter in the model eukaryotic organisms Saccharomyces cerevisiae and Caenorhabditis elegans. LuxAB expressing S. cerevisiae strains displayed distinctive rapid decays in luminescence upon addition of the bacterial luciferase substrate, n-decyl aldehyde, suggesting a toxic response. Growth studies and toxicity bioassays have subsequently confirmed, that the aldehyde substrate was toxic to both organisms at concentrations well tolerated by Escherichia coli. As the addition of aldehyde is an integral part of the bacterial luciferase activity assay, our results do not support the use of lux reporter genes for in vivo analyses in these model eukaryotic organisms.

Numerical analysis of grassland bacterial community structure under different land management regimens by using 16S ribosomal DNA sequence data and denaturing gradient gel electrophoresis banding patterns.

Bacterial diversity in unimproved and improved grassland soils was assessed by PCR amplification of bacterial 16S ribosomal DNA (rDNA) from directly extracted soil DNA, followed by sequencing of ~45 16S rDNA clones from each of three unimproved and three improved grassland samples (A. E. McCaig, L. A. Glover, and J. I. Prosser, Appl. Environ. Microbiol. 65:1721-1730, 1999) or by denaturing gradient gel electrophoresis (DGGE) of total amplification products. Semi-improved grassland soils were analyzed only by DGGE. No differences between communities were detected by calculation of diversity indices and similarity coefficients for clone data (possibly due to poor coverage). Differences were not observed between the diversities of individual unimproved and improved grassland DGGE profiles, although considerable spatial variation was observed among triplicate samples. Semi-improved grassland samples, however, were less diverse than the other grassland samples and had much lower within-group variation. DGGE banding profiles obtained from triplicate samples pooled prior to analysis indicated that there was less evenness in improved soils, suggesting that selection for specific bacterial groups occurred. Analysis of DGGE profiles by canonical variate analysis but not by principal-coordinate analysis, using unweighted data (considering only the presence and absence of bands) and weighted data (considering the relative intensity of each band), demonstrated that there were clear differences between grasslands, and the results were not affected by weighting of data. This study demonstrated that quantitative analysis of data obtained by community profiling methods, such as DGGE, can reveal differences between complex microbial communities.

Transformation of an oral bacterium via chromosomal integration of free DNA in the presence of human saliva.

Transformation of Streptococcus gordonii DL1 by free DNA was studied in human saliva. Competent S. gordonii could be transformed in vitro with plasmid DNA that had been taken into the human mouth. Transformation also occurred with a plasmid that cannot replicate in S. gordonii, but that has a region of chromosomal homology, by integration into the bacterial chromosome, although linearised plasmid DNA gave no transformants. Linear chromosomal DNA fragments did however transform S. gordonii/Tn916 efficiently in saliva when regions of homology with the recipient chromosome flanked the marker gene. These findings are discussed in relation to the potential for acquisition of DNA sequences, including genetically modified DNA, by gut and oral bacteria.

Construction of a modified mini-Tn5 luxCDABE transposon for the development of bacterial biosensors for ecotoxicity testing.

A mini-Tn5 transposon was modified to introduce a promoterless luxCDABE cassette from Vibrio fischeri into environmentally relevant bacterial strains in order to develop bioluminescence-based biosensors for toxicity testing. The mini-Tn5 luxCDABE transposon was chromosomally integrated downstream from an active promoter into two Pseudomonas strains (Pseudomonas fluorescens 8866 and Pseudomonas putida F1). Characterisation of the bioluminescent transconjugants demonstrated that the transposon integration was stable and had no effect on growth rate. Both P. fluorescens 8866 Tn5 luxCDABE and P. putida F1 Tn5 luxCDABE were used to assess the toxicity of standard solutions (Cu, Zn and 3,5-DCP) as well as Cu- and 3,5-DCP-spiked groundwater samples. They were successfully used for bioluminescence-based bioassays and the potential value of using different bacterial biosensors for ecotoxicity testing was shown.

Impact of cultivation on characterisation of species composition of soil bacterial communities.

The species composition of culturable bacteria in Scottish grassland soils was investigated using a combination of Biolog and 16S rDNA analysis for characterisation of isolates. The inclusion of a molecular approach allowed direct comparison of sequences from culturable bacteria with sequences obtained during analysis of DNA extracted directly from the same soil samples. Bacterial strains were isolated on Pseudomonas isolation agar (PIA), a selective medium, and on tryptone soya agar (TSA), a general laboratory medium. In total, 12 and 21 morphologically different bacterial cultures were isolated on PIA and TSA, respectively. Biolog and sequencing placed PIA isolates in the same taxonomic groups, the majority of cultures belonging to the Pseudomonas (sensu stricto) group. However, analysis of 16S rDNA sequences proved more efficient than Biolog for characterising TSA isolates due to limitations of the Microlog database for identifying environmental bacteria. In general, 16S rDNA sequences from TSA isolates showed high similarities to cultured species represented in sequence databases, although TSA-8 showed only 92.5% similarity to the nearest relative, Bacillus insolitus. In general, there was very little overlap between the culturable and uncultured bacterial communities, although two sequences, PIA-2 and TSA-13, showed >99% similarity to soil clones. A cloning step was included prior to sequence analysis of two isolates, TSA-5 and TSA-14, and analysis of several clones confirmed that these cultures comprised at least four and three sequence types, respectively. All isolate clones were most closely related to uncultured bacteria, with clone TSA-5.1 showing 99.8% similarity to a sequence amplified directly from the same soil sample. Interestingly, one clone, TSA-5.4, clustered within a novel group comprising only uncultured sequences. This group, which is associated with the novel, deep-branching Acidobacterium capsulatum lineage, also included clones isolated during direct analysis of the same soil and from a wide range of other sample types studied elsewhere. The study demonstrates the value of fine-scale molecular analysis for identification of laboratory isolates and indicates the culturability of approximately 1% of the total population but under a restricted range of media and cultivation conditions.

Development and application of bioluminescent Caenorhabditis elegans as multicellular eukaryotic biosensors.

We describe a novel approach to assess toxicity to the free-living nematode Caenorhabditis elegans that relies on the ability of firefly luciferase to report on endogenous ATP levels. We have constructed bioluminescent C. elegans with the luc gene under control of a constitutive promoter. Light reduction was observed in response to increasing temperature, concentrations of copper, lead and 3,5-dichlorophenol. This was due to increased mortality coupled with decreased metabolic activity in the surviving animals. The light emitted by the transgenic nematodes gave a rapid, real-time indication of metabolic status. This forms the basis of rapid and biologically relevant toxicity tests.

Design and application of a biosensor for monitoring toxicity of compounds to eukaryotes.

Here we describe an alternative approach to currently used cytotoxicity analyses through applying eukaryotic microbial biosensors. The yeast Saccharomyces cerevisiae was genetically modified to express firefly luciferase, generating a bioluminescent yeast strain. The presence of any toxic chemical that interfered with the cells' metabolism resulted in a quantitative decrease in bioluminescence. In this study, it was demonstrated that the luminescent yeast strain senses chemicals known to be toxic to eukaryotes in samples assessed as nontoxic by prokaryotic biosensors. As the cell wall and adaptive mechanisms of S. cerevisiae cells enhance stability and protect from extremes of pH, solvent exposure, and osmotic shock, these inherent properties were exploited to generate a biosensor that should detect a wide range of both organic and inorganic toxins under extreme conditions.

Chromosomal integration of the green fluorescent protein gene in lactic acid bacteria and the survival of marked strains in human gut simulations.

An integration vector was constructed to allow introduction of the gfp gene into the chromosomes of Gram-positive bacteria. Integration depends on homologous recombination between a short 458-nt sequence of the tet(M) gene in the vector and a copy of Tn916 in the host chromosome. Strains of Lactococcus lactis IL1403, Enterococcus faecalis JH2-SS, and Streptococcus gordonii DL1 stably marked with single chromosomal copies of the gfp were readily visualised by epifluorescence microscopy. The marked L. lactis strain survived poorly in a continuous culture system inoculated with human faecal flora, while the laboratory E. faecalis strain was lost at approximately the dilution rate of the fermenter.

Molecular analysis of bacterial community structure and diversity in unimproved and improved upland grass pastures.

Bacterial community structure and diversity in rhizospheres in two types of grassland, distinguished by both plant species and fertilization regimen, were assessed by performing a 16S ribosomal DNA (rDNA) sequence analysis of DNAs extracted from triplicate soil plots. PCR products were cloned, and 45 to 48 clones from each of the six libraries were partially sequenced. Phylogenetic analysis of the resultant 275 clone sequences indicated that there was considerable variation in abundance in replicate unfertilized, unimproved soil samples and fertilized, improved soil samples but that there were no significant differences in the abundance of any phylogenetic group. Several clone sequences were identical in the 16S rDNA region analyzed, and the clones comprised eight pairs of duplicate clones and two sets of triplicate clones. Many clones were found to be most closely related to environmental clones obtained in other studies, although three clones were found to be identical to culturable species in databases. The clones were clustered into operational taxonomic units at a level of sequence similarity of >97% in order to quantify diversity. In all, 34 clusters containing two or more sequences were identified, and the largest group contained nine clones. A number of diversity, dominance, and evenness indices were calculated, and they all indicated that diversity was high, reflecting the low coverage of rDNA libraries achieved. Differences in diversity between sample types were not observed. Collector's curves, however, indicated that there were differences in the underlying community structures; in particular, there was reduced diversity of organisms of the alpha subdivision of the class Proteobacteria (alpha-proteobacteria) in improved soils.

Fate of free DNA and transformation of the oral bacterium Streptococcus gordonii DL1 by plasmid DNA in human saliva.

Competitive PCR was used to monitor the survival of a 520-bp DNA target sequence from a recombinant plasmid, pVACMC1, after admixture of the plasmid with freshly sampled human saliva. The fraction of the target remaining amplifiable ranged from 40 to 65% after 10 min of exposure to saliva samples from five subjects and from 6 to 25% after 60 min of exposure. pVACMC1 plasmid DNA that had been exposed to degradation by fresh saliva was capable of transforming naturally competent Streptococcus gordonii DL1 to erythromycin resistance, although transforming activity decreased rapidly, with a half-life of approximately 50 s. S. gordonii DL1 transformants were obtained in the presence of filter-sterilized saliva and a 1-microg/ml final concentration of pVACMC1 DNA. Addition of filter-sterilized saliva instead of heat-inactivated horse serum to S. gordonii DL1 cells induced competence, although with slightly lower efficiency. These findings indicate that DNA released from bacteria or food sources within the mouth has the potential to transform naturally competent oral bacteria. However, further investigations are needed to establish whether transformation of oral bacteria can occur at significant frequencies in vivo.

Development and characterization of a lux-modified 2,4-dichlorophenol-degrading Burkholderia sp. RASC.

lux-marked biosensors for assessing the toxicity and bioremediation potential of polluted environments may complement traditional chemical techniques. luxCDABE genes were introduced into the chromosome of the 2,4-dichlorophenol (2,4-DCP)-mineralizing bacterium, Burkholderia sp. RASC c2, by biparental mating using the Tn4431 system. Experiments revealed that light output was constitutive and related to cell biomass concentration during exponential growth. The transposon insertion was stable and did not interrupt 2,4-DCP-degradative genes, and expression of luxCDABE did not constitute a metabolic burden to the cell. A bioluminescence response was detectable at sublethal 2,4-DCP concentrations: at < 10.26 microg ml(-1), bioluminescence was stimulated (e.g. 218% of control), but at concentrations >60 microg ml(-1) it declined to < 1%. Investigating the effect of [14C]-2,4-DCP concentration on the evolution of 14CO2 revealed that, for initial concentrations of 2.5-25 microg ml(-1), approximately equals 55% of the added 14C was mineralized after 24 h compared with <1% at 50 and 100 microg ml(-1). Inhibition of 2,4-DCP mineralization between 25 and 50 microg ml(-1) corresponded well to the EC50 value (33.83 microg ml(-1)) obtained from bioluminescence inhibition studies. lux-marked RASC c2 may therefore be used as a functionally (i.e. 2,4-DCP degrader) and environmentally relevant biosensor of toxicity and biodegradation inhibition.

The 3' untranslated region plays a role in the targeting of metallothionein-I mRNA to the perinuclear cytoplasm and cytoskeletal-bound polysomes.

The mechanism of localisation of metallothionein-I (MT-I) mRNA was studied in transfected cells by in situ hybridisation and cell fractionation. Hepatoma cells were transfected with the 5'-untranslated region and coding region of the beta-globin gene alone or linked to either the beta-globin 3'-untranslated region (3'-UTR) or the MT-I 3'-UTR. The wild-type beta-globin mRNA and the beta-globin mRNA lacking its native 3'-UTR were present in free and cytoskeletal-bound polysomes to a similar extent and showed no localisation. Chimaeric globin-metallothionein transcripts were significantly enriched in cytoskeletal-bound polysomes and were localised in the perinuclear cytoplasm. Chimaeric globin-metallothionein and wild-type globin transcripts were of similar stability. Chinese Hamster Ovary cells were transfected with constructs in which the MT-I 5'-untranslated region and coding sequences were linked to either the endogenous 3'-UTR or the glutathione peroxidase 3'-UTR. Wild-type MT-I transcripts were localised in the perinuclear cytoplasm but the chimaeric MT-I-glutathione peroxidase transcripts showed no distinct localisation. The results indicate that the 3'-UTR of MT-I mRNA contains a localisation signal which promotes both the association of the mRNA with the cytoskeleton and its perinuclear localisation.

Evidence for a localisation signal in the 3'-untranslated region from vimentin messenger RNA.

There is increasing evidence that some mRNAs are localised in eukaryotic somatic cells, but it is unclear what proportion of mRNAs are localised and whether this sorting involves 3'-untranslated sequences. The presence of a localisation signal within the 3'-untranslated region of vimentin mRNA was investigated by studying mRNA distribution in fibroblasts transfected with beta-globin and hybrid globin-vimentin gene constructs. In cells transfected with constructs containing either a fragment of the rabbit beta-globin gene containing both coding sequences and 3'untranslated region or the beta-globin coding sequences alone in situ hybridisation showed that beta-globin mRNA was distributed throughout the cytoplasm without any evident localisation. In contrast, in cells transfected with globin coding sequences linked to the vimentin 3'-untranslated region there was a strong perinuclear localisation of the hybrid mRNA. The results show that loss of its endogenous 3'-untranslated region does not affect distribution of beta-globin mRNA whereas the vimentin 3'-untranslated region causes an altered localisation of beta-globin mRNA. We conclude that the vimentin 3'-untranslated region contains a localisation signal which can direct reporter sequences to the perinuclear cytoplasm.

Cell density-regulated recovery of starved biofilm populations of ammonia-oxidizing bacteria.

The speed of recovery of cell suspensions and biofilm populations of the ammonia oxidizer Nitrosomonas europaea, following starvation was determined. Stationary-phase cells, washed and resuspended in ammoniumfree inorganic medium, were starved for periods of up to 42 days, after which the medium was supplemented with ammonium and subsequent growth was monitored by measuring nitrite concentration changes. Cultures exhibited a lag phase prior to exponential nitrite production, which increased from 8.72 h (no starvation) to 153 h after starvation for 42 days. Biofilm populations of N. europaea colonizing sand or soil particles in continuous-flow, fixed column reactors were starved by continuous supply of ammonium-free medium. Following resupply of ammonium, starved biofilms exhibited no lag phase prior to nitrite production, even after starvation for 43.2 days, although there was evidence of cell loss during starvation. Biofilm formation will therefore provide a significant ecological advantage for ammonia oxidizers in natural environments in which the substrate supply is intermittent. Cell density-dependent phenomena in a number of gram-negative bacteria are mediated by N-acyl homoserine lactones (AHL), including N-(3-oxohexanoyl)-L-homoserine lactone (OHHL). Addition of both ammonium and OHHL to cell suspensions starved for 28 days decreased the lag phase in a concentration-dependent manner from 53.4 h to a minimum of 10.8 h. AHL production by N. europaea was detected by using a luxR-luxAB AHL reporter system. The results suggest that rapid recovery of high-density biofilm populations may be due to production and accumulation of OHHL to levels not possible in relatively low-density cell suspensions.