Angelika Lehner

Deciphering the evolution and metabolism of an anammox bacterium from a community genome

Anaerobic ammonium oxidation (anammox) has become a main focus in oceanography and wastewater treatment. It is also the nitrogen cycles major remaining biochemical enigma. Among its features, the occurrence of hydrazine as a free intermediate of catabolism, the biosynthesis of ladderane lipids and the role of cytoplasm differentiation are unique in biology. Here we use environmental genomics—the reconstruction of genomic data directly from the environment—to assemble the genome of the uncultured anammox bacterium Kuenenia stuttgartiensis from a complex bioreactor community. The genome data illuminate the evolutionary history of the Planctomycetes and allow us to expose the genetic blueprint of the organisms special properties. Most significantly, we identified candidate genes responsible for ladderane biosynthesis and biological hydrazine metabolism, and discovered unexpected metabolic versatility.

Oligonucleotide Microarray for 16S rRNA Gene-Based Detection of All Recognized Lineages of Sulfate-Reducing Prokaryotes in the Environment

ABSTRACT For cultivation-independent detection of sulfate-reducing prokaryotes (SRPs) an oligonucleotide microarray consisting of 132 16S rRNA gene-targeted oligonucleotide probes (18-mers) having hierarchical and parallel (identical) specificity for the detection of all known lineages of sulfate-reducing prokaryotes (SRP-PhyloChip) was designed and subsequently evaluated with 41 suitable pure cultures of SRPs. The applicability of SRP-PhyloChip for diversity screening of SRPs in environmental and clinical samples was tested by using samples from periodontal tooth pockets and from the chemocline of a hypersaline cyanobacterial mat from Solar Lake (Sinai, Egypt). Consistent with previous studies, SRP-PhyloChip indicated the occurrence of Desulfomicrobium spp. in the tooth pockets and the presence of Desulfonema- and Desulfomonile-like SRPs (together with other SRPs) in the chemocline of the mat. The SRP-PhyloChip results were confirmed by several DNA microarray-independent techniques, including specific PCR amplification, cloning, and sequencing of SRP 16S rRNA genes and the genes encoding the dissimilatory (bi)sulfite reductase (dsrAB).

Cronobacter gen. nov., a new genus to accommodate the biogroups of Enterobacter sakazakii, and proposal of Cronobacter sakazakii gen. nov., comb. nov., Cronobacter malonaticus sp. nov., Cronobacter turicensis sp. nov., Cronobacter muytjensii sp. nov., Cronobacter dublinensis sp. nov., Cronobacter genomospecies 1, and of three subspecies, Cronobacter dublinensis subsp. dublinensis subsp. nov., Cronobacter dublinensis subsp. lausannensis subsp. nov. and Cronobacter dublinensis subsp. lactaridi subsp. nov.

[Enterobacter] sakazakii is an opportunistic pathogen that can cause infections in neonates. This study further clarifies the taxonomy of isolates described as [E.] sakazakii and completes the formal description of the proposed reclassification of these organisms as novel species and subspecies within a proposed novel genus, Cronobacter gen. nov. [E.] sakazakii was first defined in 1980, however recent polyphasic taxonomic analysis has determined that this group of organisms consists of several genomospecies. In this study, the phenotypic descriptions of the proposed novel species are expanded using Biotype 100 and Biolog Phenotype MicroArray data. Further DNA-DNA hybridization experiments showed that malonate-positive strains within the [E.] sakazakii genomospecies represent a distinct species, not a subspecies. DNA-DNA hybridizations also determined that phenotypically different strains within the proposed species, Cronobacter dublinensis sp. nov., belong to the same species and can be considered as novel subspecies. Based on these analyses, the following alternative classifications are proposed: Cronobacter sakazakii gen. nov., comb. nov. [type strain ATCC 29544(T) (=NCTC 11467(T))]; Cronobacter malonaticus sp. nov. [type strain CDC 1058-77(T) (=LMG 23826(T)=DSM 18702(T))]; Cronobacter turicensis sp. nov. [type strain z3032(T) (=LMG 23827(T)=DSM 18703(T))]; Cronobacter muytjensii sp. nov. [type strain ATCC 51329(T) (=CIP 103581(T))]; Cronobacter dublinensis sp. nov. [type strain DES187(T) (=LMG 23823(T)=DSM 18705(T))]; Cronobacter dublinensis subsp. dublinensis subsp. nov. [type strain DES187(T) (=LMG 23823(T)=DSM 18705(T))]; Cronobacter dublinensis subsp. lausannensis subsp. nov. [type strain E515(T) (=LMG 23824=DSM 18706(T))], and Cronobacter dublinensis subsp. lactaridi subsp. nov. [type strain E464(T) (=LMG 23825(T)=DSM 18707(T))].

16S rRNA Gene-Based Oligonucleotide Microarray for Environmental Monitoring of the Betaproteobacterial Order “Rhodocyclales”

ABSTRACT For simultaneous identification of members of the betaproteobacterial order “Rhodocyclales” in environmental samples, a 16S rRNA gene-targeted oligonucleotide microarray (RHC-PhyloChip) consisting of 79 probes was developed. Probe design was based on phylogenetic analysis of available 16S rRNA sequences from all cultured and as yet uncultured members of the “Rhodocyclales.” The multiple nested probe set was evaluated for microarray hybridization with 16S rRNA gene PCR amplicons from 29 reference organisms. Subsequently, the RHC-PhyloChip was successfully used for cultivation-independent “Rhodocyclales” diversity analysis in activated sludge from an industrial wastewater treatment plant. The implementation of a newly designed “Rhodocyclales”-selective PCR amplification system prior to microarray hybridization greatly enhanced the sensitivity of the RHC-PhyloChip and thus enabled the detection of “Rhodocyclales” populations with relative abundances of less than 1% of all bacteria (as determined by fluorescence in situ hybridization) in the activated sludge. The presence of as yet uncultured Zoogloea-, Ferribacterium/Dechloromonas-, and Sterolibacterium-related bacteria in the industrial activated sludge, as indicated by the RHC-PhyloChip analysis, was confirmed by retrieval of their 16S rRNA gene sequences and subsequent phylogenetic analysis, demonstrating the suitability of the RHC-PhyloChip as a novel monitoring tool for environmental microbiology.

The Isotope Array, a New Tool That Employs Substrate-Mediated Labeling of rRNA for Determination of Microbial Community Structure and Function

ABSTRACT A new microarray method, the isotope array approach, for identifying microorganisms which consume a 14C-labeled substrate within complex microbial communities was developed. Experiments were performed with a small microarray consisting of oligonucleotide probes targeting the 16S rRNA of ammonia-oxidizing bacteria (AOB). Total RNA was extracted from a pure culture of Nitrosomonas eutropha grown in the presence of [14C]bicarbonate. After fluorescence labeling of the RNA and microarray hybridization, scanning of all probe spots for fluorescence and radioactivity revealed that specific signals were obtained and that the incorporation of 14C into rRNA could be detected unambiguously. Subsequently, we were able to demonstrate the suitability of the isotope array approach for monitoring community composition and CO2 fixation activity of AOB in two nitrifying activated-sludge samples which were incubated with [14C]bicarbonate for up to 26 h. AOB community structure in the activated-sludge samples, as predicted by the microarray hybridization pattern, was confirmed by quantitative fluorescence in situ hybridization (FISH) and comparative amoA sequence analyses. CO2 fixation activities of the AOB populations within the complex activated-sludge communities were detectable on the microarray by 14C incorporation and were confirmed independently by combining FISH and microautoradiography. AOB rRNA from activated sludge incubated with radioactive bicarbonate in the presence of allylthiourea as an inhibitor of AOB activity showed no incorporation of 14C and thus was not detectable on the radioactivity scans of the microarray. These results suggest that the isotope array can be used in a PCR-independent manner to exploit the high parallelism and discriminatory power of microarrays for the direct identification of microorganisms which consume a specific substrate in the environment.

Comparison of Different Approaches To Quantify Staphylococcus aureus Cells by Real-Time Quantitative PCR and Application of This Technique for Examination of Cheese

ABSTRACT Two different real-time quantitative PCR (RTQ-PCR) approaches were applied for PCR-based quantification of Staphylococcus aureus cells by targeting the thermonuclease (nuc) gene. Purified DNA extracts from pure cultures ofS. aureus were quantified in a LightCycler system using SYBR Green I. Quantification proved to be less sensitive (60nuc gene copies/μl) than using a fluorigenic TaqMan probe (6 nuc gene copies/μl). Comparison of the LightCycler system and the well-established ABI Prism 7700 SDS with TaqMan probes revealed no statistically significant differences with respect to sensitivity and reproducibility. Application of the RTQ-PCR assay to quantify S. aureus cells in artificially contaminated cheeses of different types achieved sensitivities from 1.5 × 102 to 6.4 × 102 copies of the nuc gene/2 g, depending on the cheese matrix. The coefficients of correlation between log CFU and nuc gene copy numbers ranged from 0.979 to 0.998, thus enabling calculation of the number of CFU of S. aureus in cheese by performing RTQ-PCR.

Microarray and Functional Gene Analyses of Sulfate-Reducing Prokaryotes in Low-Sulfate, Acidic Fens Reveal Cooccurrence of Recognized Genera and Novel Lineages

ABSTRACT Low-sulfate, acidic (approximately pH 4) fens in the Lehstenbach catchment in the Fichtelgebirge mountains in Germany are unusual habitats for sulfate-reducing prokaryotes (SRPs) that have been postulated to facilitate the retention of sulfur and protons in these ecosystems. Despite the low in situ availability of sulfate (concentration in the soil solution, 20 to 200 μM) and the acidic conditions (soil and soil solution pHs, approximately 4 and 5, respectively), the upper peat layers of the soils from two fens (Schlöppnerbrunnen I and II) of this catchment displayed significant sulfate-reducing capacities. 16S rRNA gene-based oligonucleotide microarray analyses revealed stable diversity patterns for recognized SRPs in the upper 30 cm of both fens. Members of the family “Syntrophobacteraceae” were detected in both fens, while signals specific for the genus Desulfomonile were observed only in soils from Schlöppnerbrunnen I. These results were confirmed and extended by comparative analyses of environmentally retrieved 16S rRNA and dissimilatory (bi)sulfite reductase (dsrAB) gene sequences; dsrAB sequences from Desulfobacca-like SRPs, which were not identified by microarray analysis, were obtained from both fens. Hypotheses concerning the ecophysiological role of these three SRP groups in the fens were formulated based on the known physiological properties of their cultured relatives. In addition to these recognized SRP lineages, six novel dsrAB types that were phylogenetically unrelated to all known SRPs were detected in the fens. These dsrAB sequences had no features indicative of pseudogenes and likely represent novel, deeply branching, sulfate- or sulfite-reducing prokaryotes that are specialized colonists of low-sulfate habitats.

Detection and quantification of the iap gene of Listeria monocytogenes and Listeria innocua by a new real-time quantitative PCR assay

A real-time quantitative polymerase chain reaction (PCR) assay for direct detection and enumeration of Listeria monocytogenes and Listeria innocua was developed and applied to artificially contaminated milk samples. The iap gene present in both species was used as a target for amplification of a 175-bp (L. monocytogenes) and a 309-bp (L. innocua) fragment. To ensure that L. monocytogenes and L. innocua are specifically detectable, tests were carried out using 42 L. monocytogenes strains and 33 L. innocua strains belonging to different serovars. Specificity was also confirmed using 22 bacterial strains not belonging to the genus Listeria, including closely related bacteria. In addition to specificity, the reported assay is characterized by a wide dynamic range of quantification and a high sensitivity, as we could detect as few as six copies of the iap gene per PCR using purified DNA as template. When applied to direct detection and quantification of L. monocytogenes in milk, the more rapid real-time quantitative PCR assay was as sensitive as the traditional plate count method, but real-time quantitative PCR-derived iap gene copy numbers were one to two logs higher than colony-forming units obtained by the plate count method.

Biofilm formation, extracellular polysaccharide production, and cell-to-cell signaling in various Enterobacter sakazakii strains: aspects promoting environmental persistence.

Enterobacter sakazakii is considered an opportunistic pathogen and has been implicated in food-associated cases of meningitis or enteritis, especially in neonates and infants. The organism has been detected in various types of food and in food production units, but so far only powdered infant formula has been linked to outbreaks of disease. Survival and persistence in such environments requires the ability to adapt to high osmotic potentials and/or dry conditions. Fifty-six E. sakazakii strains were evaluated for several features important for persistence and survival: (i) biofilm formation and the putative production of cellulose as one of the components of the extracellular matrix, (ii) adherence to hydrophilic and hydrophobic surfaces, (iii) the production of extracellular polysaccharides, and (iv) the ability of E. sakazakii to produce cell-to-cell signaling molecules. Pellicle and flock formation was observed in 21 of the strains grown in Luria-Bertani broth and in 44 of the strains grown in brain heart infusion broth. Calcofluor-stained fibrils, observed microscopically in every (fragile or rigid) pellicle, suggested the presence of cellulose as an extracellular compound in this type of biofilm. Twelve isolates did not form any pellicle or flocks under either condition. Twenty-three of the isolates exhibited the potential to adhere to glass surfaces in shaken cultures, and 33 strains showed biofilm formation at the air-solid interface of polyvinyl chloride microtiter wells. Sixteen isolates adhered to both surfaces. Twenty-four of the isolates tested produced a milky, viscous mass, considered as extracellular polysaccharide. High-performance liquid chromatography analysis of the polysaccharide revealed the presence of glucose, galactose, fucose, and glucuronic acid. Thin-layer chromatography analyses performed on ethyl acetate extracts of cell-free supernatants of the 56 strains indicated the presence of two different types of acylated homoserine lactones (3-oxo-C6-HSL and 3-oxo-C8-HSL). These findings illustrate the ability of E. sakazakii to produce cell-to-cell signaling molecules.

Microbiological, Epidemiological, and Food Safety Aspects of Enterobacter sakazakii

Enterobacter sakazakii is considered to be an opportunistic pathogen and has been implicated in foodborne diseases causing meningitis or enteritis, especially in neonates and infants. The U.S FoodNet 2002 survey rate of invasive infections with this organism in infants under 1 year of age was 1 per 100,000 infants. Severity of the disease is a matter of concern. In a recent study on the occurrence of E. sakazakii in production environments from food (milk powder, chocolate, cereal, potato, and pasta) factories and households, this organism was isolated with varying frequency from nearly all environments examined, strongly indicating that it is widespread. Stationary phase E. sakazakii cells are remarkably resistant to osmotic and drying stresses compared with other species of the Enterobacteriacae. In this article, we review the literature on this organism with special respect to the information relevant for food safety.