Genotyping of environmental and clinical Stenotrophomonas maltophilia isolates and their pathogenic potential.

Stenotrophomonas maltophilia is a highly versatile species with useful biotechnological potential but also with pathogenic properties. In light of possible differences in virulence characteristics, knowledge about genomic subgroups is therefore desirable. Two different genotyping methods, rep-PCR fingerprinting and partial gyrB gene sequencing were used to elucidate S. maltophilia intraspecies diversity. Rep-PCR fingerprinting revealed the presence of 12 large subgroups, while gyrB gene sequencing distinguished 10 subgroups. For 8 of them, the same strain composition was shown with both typing methods. A subset of 59 isolates representative for the gyrB groups was further investigated with regards to their pathogenic properties in a virulence model using Dictyostelium discoideum and Acanthamoeba castellanii as host organisms. A clear tendency towards accumulation of virulent strains could be observed for one group with A. castellanii and for two groups with D. discoideum. Several virulent strains did not cluster in any of the genetic groups, while other groups displayed no virulence properties at all. The amoeba pathogenicity model proved suitable in showing differences in S. maltophilia virulence. However, the model is still not sufficient to completely elucidate virulence as critical for a human host, since several strains involved in human infections did not show any virulence against amoeba.

Plasmid-mediated bioaugmentation of wastewater microbial communities in a laboratory-scale bioreactor.

Xenobiotic degradation during biological wastewater treatment can be established or enhanced by bioaugmentation - the addition of biological agents carrying biodegradation genes required to perform the task. Whereas the addition of microbial cells carrying chromosomally encoded catabolic genes can be impaired by limited survival of the added microorganisms, the addition of donor organisms carrying a transmissible catabolic plasmid is a promising alternative. This plasmid can spread within the indigenous microbial community of the system, circumventing the need for extended survival of the introduced bacterial strain. Here we discuss how the catabolic plasmid pNB2 can be evaluated towards its potential to facilitate the degradation of a xenobiotic compound, 3-chloroaniline, and demonstrate the applicability of this plasmid to accomplish 3-chloroaniline degradation in a bioreactor setting after in situ transfer to suitable recipient strains.

Nitrification in fixed-bed reactors treating saline wastewater.

Halophilic nitrifiers belonging to the genus Nitrosomonas and Nitrospira were enriched from seawater and marine sediment samples of the North Sea. The maximal ammonia oxidation rate (AOR) in batch enrichments with seawater was 15.1 mg N L(-1) day(-1). An intermediate nitrite accumulation was observed. Two fixed-bed reactors for continuous nitrification with either polyethylene/claysinter lamellas (FBR A) or porous ceramic rings (FBR B)were run at two different ammonia concentrations, three different ammonia loading rates (ALRs), + or - pH adjustment,and at an increased upflow velocity. A better overall nitrification without nitrite accumulation was observed in FBR B. However, FBR A revealed a higher AOR and nitrite oxidation rate of 6 and 7 mg N L(-1) h(-1), compared to FBR B with 5 and 5.9 mg N L(-1) h(-1), respectively. AORs in the FBRs were at least ten times higher than in suspended enrichment cultures. Whereas a shift within the ammoniaoxidizing population in the genus Nitrosomonas at the subspecies level occurred in FBR B with synthetic seawater at an increasing ALR and a decreasing pH, the nitrite oxidizing Nitrospira population apparently did not change.

Pseudorhodoferax soli gen. nov., sp. nov. and Pseudorhodoferax caeni sp. nov., two members of the class Betaproteobacteria belonging to the family Comamonadaceae.

A novel betaproteobacterium, strain TBEA3(T), was isolated from soil using enrichment cultures with the organic thioether 3,3'-thiodipropionic acid as sole carbon and energy source. Analysis of the 16S rRNA gene sequence revealed 99.1 % sequence similarities to a poorly characterized member of the family Comamonadaceae, strain SB1(T), which had been previously isolated from activated sludge. Both strains showed highest gene sequence similarities (up to 96.9 %) to members of the genera Rhodoferax and Curvibacter. The DNA G+C contents of strains TBEA3(T) and SB1(T) were 69.1 and 70.1 mol%, respectively, and the DNA-DNA hybridization value between these two strains was 45.3 %. The predominant cellular fatty acids in both strains were C(16 : 0), C(18 : 1)omega7c and summed feature 3 (C(16 : 1)omega7c and/or C(15 : 0) iso 2-OH). The major 3-hydroxy fatty acid was C(10 : 0) 3-OH. Based on the genetic and chemotaxonomic data, strains TBEA3(T) and SB1(T) represent two novel species of a new genus within the family Comamonadaceae, for which the name Pseudorhodoferax gen. nov. is proposed. Strain TBEA3(T) (=LMG 24555(T)=DSM 21634(T)) is assigned to Pseudorhodoferax soli sp. nov., as the type strain of the type species of the genus. Strain SB1(T) (=LMG 24543(T)=DSM 21598(T)) is the type strain of Pseudorhodoferax caeni sp. nov.

Ferrous iron oxidation and rusticyanin in halotolerant, acidophilic 'Thiobacillus prosperus'.

The halotolerant acidophile 'Thiobacillus prosperus' was shown to require chloride for growth. With ferrous iron as substrate, growth occurred at a rate similar to that of the well-studied acidophile Acidithiobacillus ferrooxidans. Previously, the salt (NaCl) requirement of 'T. prosperus' was not clear and its growth on ferrous iron was described as poor. A subtractive hybridization of cDNAs from ferrous-iron-grown and sulfur-grown 'T. prosperus' strain V6 led to identification of a cluster of genes similar to the rus operon reported to encode ferrous iron oxidation in A. ferrooxidans. However, the 'T. prosperus' gene cluster did not contain a homologue of cyc1, which is thought to encode a key cytochrome c in the pathway of electron transport from ferrous iron in A. ferrooxidans. Rusticyanin, another key protein in ferrous iron oxidation by A. ferrooxidans, was present in 'T. prosperus' at similar concentrations in cells grown on either ferrous iron or sulfur.

Bioaugmentation of activated sludge towards 3-chloroaniline removal with a mixed bacterial population carrying a degradative plasmid.

A bioaugmentation approach combining several strategies was applied to achieve degradation of 3-chloroaniline (3CA) in semicontinuous activated sludge reactors. In a first step, a 3CA-degrading Comamonas testosteroni strain carrying the degradative plasmid pNB2 was added to a biofilm reactor, and complete 3CA degradation together with spread of the plasmid within the indigenous biofilm population was achieved. A second set of reactors was then bioaugmented with either a suspension of biofilm cells removed from the carrier material or with biofilm-containing carrier material. 3CA degradation was established rapidly in all bioaugmented reactors, followed by a slow adaptation of the non-bioaugmented control reactors. In response to variations in 3CA concentration, all reactors exhibited temporary performance breakdowns. Whereas duplicates of the control reactors deviated in their behaviour, the bioaugmented reactors appeared more reproducible in their performance and population dynamics. Finally, the carrier-bioaugmented reactors showed an improved performance in the presence of high 3CA influent concentrations over the suspension-bioaugmented reactors. In contrast, degradation in one control reactor failed completely, but was rapidly established in the remaining control reactor.

Ferrous iron- and sulfur-induced genes in Sulfolobus metallicus.

Genes of Sulfolobus metallicus that appeared to be upregulated in relation to growth on either ferrous iron or sulfur were identified using subtractive hybridization of cDNAs. The genes upregulated during growth on ferrous iron were found in a cluster, and most were predicted to encode membrane proteins. Quantitative reverse transcription-PCR of cDNA showed upregulation of most of these genes during growth on ferrous iron and pyrite compared to results during growth on sulfur. The highest expression levels observed included those for genes encoding proteins with similarities to cytochrome c oxidase subunits and a CbsA-like cytochrome. The genes identified here that may be involved in oxidation of ferrous iron by S. metallicus are termed fox genes. Of three available genomes of Sulfolobus species (S. tokodaii, S. acidocaldarius, and S. solfataricus), only that of S. tokodaii has a cluster of highly similar open reading frames, and only S. tokodaii of these three species was also able to oxidize ferrous iron. A gene encoding sulfur oxygenase-reductase was identified as the source of the dominant transcript in sulfur-grown cells of S. metallicus, with the predicted protein showing high identities to the previously described examples from S. tokodaii and species of Acidianus.

Design and evaluation of 16S rRNA sequence based oligonucleotide probes for the detection and quantification of Comamonas testosteroni in mixed microbial communities.

BACKGROUND: The beta-proteobacterial species Comamonas testosteroni is capable of biotransformation and also biodegradation of a range of chemical compounds and thus potentially useful in chemical manufacturing and bioremediation. The ability to detect and quantify members of this species in mixed microbial communities thus may be desirable. RESULTS: We have designed an oligonucleotide probe for use in fluorescent in situ hybridization (FISH) and two pairs of PCR primers targeting a C. testosteroni subgroup. The FISH probe and one of the PCR primer pairs are suitable for quantification of C. testosteroni in mixed microbial communities using FISH followed by quantitative image analysis or real-time quantitative PCR, respectively. This has been shown by analysis of samples from an enrichment of activated sludge on testosterone resulting in an increase in abundance and finally isolation of C. testosteroni. Additionally, we have successfully used quantitative PCR to follow the C. testosteroni abundance during a laboratory scale wastewater bioaugmentation experiment. CONCLUSION: The oligonucleotides presented here provide a useful tool to study C. testosteroni population dynamics in mixed microbial communities.

Genetic and phenotypic microdiversity of Ochrobactrum spp.

The diversity of Ochrobactrum anthropi, Ochrobactrum intermedium, Ochrobactrum tritici and Ochrobactrum grignonense in agricultural soil and on the wheat rhizoplane was investigated. O. anthropi was isolated both from soil and from the rhizoplane, O. intermedium and grignonense only from bulk soil, and O. tritici only from the wheat rhizoplane. On the genetic level, the immunotrapped isolates and a number of strains from culture collection mainly of clinical origin were compared with rep-PCR profiling using BOX primers, and a subset of these isolates and strains using REP primers. The isolates clustered according to their species affiliation. There was no correlation between rep clusters of O. anthropi isolates and habitat (place of isolation). The genetic diversity of Ochrobactrum at the species level as well as microdiversity of O. anthropi (number of BOX groups) was higher in soil than on the rhizoplane. Similarity values from genetic rep-PCR profiles correlated positively with DNA-DNA reassociation percentages. Isolates with >80.7% similarity in BOX profile and >86.4% in rep profile clustered within the same species. Similarity analysis of rep-PCR profiles is hence an alternative to DNA-DNA hybridization as a genomic criterion for species delineation within the genus Ochrobactrum. We used the substrate utilization system BIOLOG-GN to compare the immunotrapped isolates on the phenetic level. For the isolates from bulk soil, substrate utilization versatility (number of utilized substrates) and substrate utilization capacity (mean conversion rate of substrates) were slightly but significantly higher than for the isolates from the rhizoplane. This trend was also seen using API 20E and 20NE systems. Plate counts of total bacteria and the number of immunotrapped Ochrobactrum isolates per gram dry weight were higher for the rhizoplane than for the soil samples. The results of genetic and phenotypic analyses indicated a 'rhizosphere effect'; the diversity and metabolic capacity of Ochrobactrum isolates were higher in bulk soil, and the population density was higher on the wheat rhizoplane.

Comparative sequence analysis of the internal transcribed spacer 1 of Ochrobactrum species.

The internal 16S/23S rDNA (rrs/rrl) internal spacer region 1 (ITS1) of 54 Ochrobactrum strains and close relatives was analysed. Separation of ITS1 containing PCR products by gel-electrophoresis, DGGE, cloning and sequencing revealed ITS1 length and sequence heterogeneity. We found up to 5 different allelic ITS1 stretches within a single strain (Ochrobactrum intermedium LMG 3301T), and 2-3 different ITS1 alleles in O. tritici. Within ITS1, ITS1c, being part of the conserved double-stranded rrn processing stem dsPS1, produced the most reliable segment tree. The overall ITS1, ITS1c and rrs phylogenetic tree topologies were generally consistent, but there was evidence for horizontal rrn (segment) transfer in O. tritici LMG 2134 (formerly O. anthropi). Good correlations were found between ITS1, ITS1c and rrs sequence similarity and DNA-DNA hybridization values indicating that phylogenetic analysis of ITS1 and ITS1c both can be used to preliminarily deduce the phylogenetic affiliation if HGT was excluded. Strains sharing > 96.19% ITS1c (> 95.11% ITS1) similarity fell within a species, and < or = 68.42% ITS1c (< or = 70.33% ITS1) similarity outside a genus. Both ITS1 and ITS1c analysis resolved microdiversity more profoundly than rrs analysis and revealed clades (genomovars) within O. anthropi that were also produced in rep cluster analysis. There was no evidence for habitat-specific ITS1 genomovars within Ochrobactrum species. Diversity of Ochrobactrum was higher in soil than at the rhizoplane below and at the species level. Isolates from soil contained only 1 rrn type whereas isolates from human clinical, animal and rhizoplane specimens could contain more.

High phylogenetic diversity of transconjugants carrying plasmid pJP4 in an activated sludge-derived microbial community.

Transconjugants of plasmid pJP4, originating from an agar plate mating of a Pseudomonas putida donor with an activated sludge-derived microbial community, were isolated and identified by partial 16S rDNA sequencing. The transconjugant strains belonged to a variety of genera of the alpha-, beta-, gamabeta- and gamma-classes of the Proteobacteria, mostly to the families Rhizobiaceae and Comamonadaceae and the genus Stenotrophomonas. Only P. putida and Delftia spp. strains were able to grow on 2,4-D as the sole carbon source.

Dual labeling of Pseudomonas putida with fluorescent proteins for in situ monitoring of conjugal transfer of the TOL plasmid.

We describe here a dual-labeling technique involving the green fluorescent protein (GFP) and the red fluorescent protein (DsRed) for in situ monitoring of horizontal gene transfer via conjugation. A GFPmut3b-tagged derivative of narrow-host-range TOL plasmid (pWWO) was delivered to Pseudomonas putida KT2442, which was chromosomally labeled with dsRed by transposon insertion via biparental mating. Green and red fluorescent proteins were coexpressed in donor P. putida cells. Cells expressing both fluorescent proteins were smaller in size than cells expressing GFP alone. Donors and transconjugants in mixed culture or sludge samples were discriminated on the basis of their fluorescence by using confocal laser scanning microscopy. Conjugal plasmid transfer frequencies on agar surfaces and in sludge microcosms were determined microscopically without cultivation. This method worked well for in situ monitoring of horizontal gene transfer in addition to tracking the fate of microorganisms released into complex environments. To the best of our knowledge, this is the first study that discusses the coexpression of GFP and DsRed for conjugal gene transfer studies.

Mechanism of sulfide-quinone reductase investigated using site-directed mutagenesis and sulfur analysis.

Biological sulfide oxidation is a reaction occurring in all three domains of life. One enzyme responsible for this reaction in many bacteria has been identified as sulfide:quinone oxidoreductase (SQR). The enzyme from Rhodobacter capsulatus is a peripherally membrane-bound flavoprotein with a molecular mass of approximately 48 kDa, presumably acting as a homodimer. In this work, SQR from Rb. capsulatus has been modified with an N-terminal His tag and heterologously expressed in and purified from Escherichia coli. Three cysteine residues have been shown to be essential for the reductive half-reaction by site-directed mutagenesis. The catalytic activity has been nearly completely abolished after mutation of each of the cysteines to serine. A decrease in fluorescence on reduction by sulfide as observed for the wild-type enzyme has not been observed for any of the mutated enzymes. Mutation of a conserved valine residue to aspartate within the third flavin-binding domain led to a drastically reduced substrate affinity, for both sulfide and quinone. Two conserved histidine residues have been mutated individually to alanine. Both of the resulting enzymes exhibited a shift in the pH dependence of the SQR reaction. Polysulfide has been identified as a primary reaction product using spectroscopic and chromatographic methods. On the basis of these data, reaction mechanisms for sulfide-dependent reduction and quinone-dependent oxidation of the enzyme and for the formation of polysulfide are proposed.