Klaus Neuhaus

Remote control of tumour-targeted Salmonella enterica serovar Typhimurium by the use of L-arabinose as inducer of bacterial gene expression in vivo.

We have used Salmonella enterica serovar Typhimurium (S. typhimurium) which are able to colonize tumours besides spleen and liver. Bacteria were equipped with constructs encoding green fluorescent protein or luciferase as reporters under control of the promoter PBAD that is inducible with l‐arabinose. Reporter genes could be induced in culture but also when the bacteria resided within the mouse macrophages J774A.1. More important, strong expression of reporters by the bacteria could be detected in mice after administration of l‐arabinose. This was especially pronounced in bacteria colonizing tumours. Histology demonstrated that the bacteria had accumulated in and close to necrotic areas of tumours. Bacterial gene induction was observed in both regions. PBAD is tightly controlled also in vivo because gene E of bacteriophage ΦX174 could be introduced as inducible suicide gene. The possibility to deliberately induce genes in bacterial carriers within the host should render them extremely powerful tools for tumour therapy.

Listeria weihenstephanensis sp. nov., isolated from the water plant Lemna trisulca taken from a freshwater pond.

The phylogenetic position and phenotypic characteristics of two non-spore-forming bacilli similar to members of the genus Listeria were studied. The gram-reaction-positive, slightly motile, facultatively anaerobic strains were isolated from the water plant Lemna trisulca sampled from a freshwater pond in Bavaria, Germany. Although no identification was possible employing the API Listeria test (bioMérieux), 16S rRNA sequence analysis confirmed a close phylogenetic similarity to Listeria rocourtiae DSM 22097(T) (99.0 % sequence similarity) and a more distant relationship to other Listeria species (96.0 % to Listeria monocytogenes DSM 20600(T) and 95.0 % similarity to Listeria grayi DSM 20601(T)). DNA-DNA hybridization analysis between the isolates and Listeria rocourtiae DSM 22097(T) yielded a similarity of 22.5 %. Analysis of partial sequences of sigB, prs, recA and HSP60 were studied and compared with those of other members of the genus Listeria and Brochothrix thermosphacta DSM 20171(T) supporting the relationships indicated by 16S rRNA gene sequences. The studied isolates were non-haemolytic and were not associated with cases of human or animal disease. While the results demonstrate that the strains belong to the genus Listeria, phenotypic and genotypic differences from Listeria rocourtiae DSM 22097(T) suggest that the strains represent a novel species for which the name Listeria weihenstephanensis sp. nov. is proposed; the type strain is WS 4560(T) ( = DSM 24698(T) = LMG 26374(T)), with WS 4615 ( = DSM 24699 = LMG 26375) as a second strain of the species.

Rapid discrimination of psychrotolerant and mesophilic strains of the Bacillus cereus group by PCR targeting of 16S rDNA

The paper describes a novel PCR assay for discriminating psychrotolerant and mesophilic strains of the Bacillus cereus group by targeting of 16S rDNA signatures. Application of the assay circumvents long-term growth tests at low temperature currently used to detect psychrotolerant strains. PCR was performed with pure cultures. A 100% correlation of PCR and growth data at 7°C was obtained for the 194 B. cereus group strains tested. Potential applications of the assay for the dairy industry and agriculture are suggested.

Restart of Exponential Growth of Cold-Shocked Yersinia enterocolitica Occurs after Down-Regulation of cspA1/A2 mRNA

The cellular content of major cold shock protein (MCSP) mRNA transcribed from the tandem gene duplication cspA1/A2 and growth of Yersinia enterocolitica were compared when exponentially growing cultures of this bacterium were cold shocked from 30 to 20, 15, 10, 5, or 0 degrees C, respectively. A clear correlation between the time point when exponential growth resumes after cold shock and the degradation of cspA1/A2 mRNA was found. A polynucleotide phosphorylase-deficient mutant was unable to degrade cspA1/A2 mRNA properly and showed a delay, as well as a lower rate, of growth after cold shock. For this mutant, a correlation between decreasing cspA1/A2 mRNA and restart of growth after cold shock was also observed. For both wild-type and mutant cells, no correlation of restart of growth with the cellular content of MCSPs was found. We suggest that, after synthesis of cold shock proteins and cold adaptation of the cells, MCSP mRNAs must be degraded; otherwise, they trap ribosomes, prevent translation of bulk mRNA, and thus inhibit growth of this bacterium at low temperatures.

Transcriptional Analysis of Long-Term Adaptation of Yersinia enterocolitica to Low-Temperature Growth

To analyze the transcriptional response of Yersinia enterocolitica cells to prolonged growth at low temperature, a collection of luxCDABE transposon mutants was cultivated in parallel at optimal (30 degrees C) and suboptimal (10 degrees C) temperatures and screened for enhanced promoter activities during growth until entering stationary phase. Among 5,700 Y. enterocolitica mutants, 42 transcriptional units were identified with strongly enhanced or reduced promoter activity at 10 degrees C compared to 30 degrees C, and changes in their transcriptional levels over time were measured. Green fluorescent protein fusions to 10 promoter regions confirmed the data. The temporal order of induction of the temperature-responsive genes of Y. enterocolitica was deduced, starting with the expression of cold shock genes cspA and cspB and the elevated transcription of a glutamate-aspartate symporter. Subsequently, cold-adapted cells drastically up-regulated genes encoding environmental sensors and regulators, such as UhpABC, ArcA, and methyl-accepting chemotaxis protein I (MCPI). Among the most prominent cold-responsive elements that were transcriptionally induced during growth in early and middle exponential phase are the insecticidal toxin genes tcaA and tcaB, as well as genes involved in flagellar synthesis and chemotaxis. The expression pattern of the late-exponential- to early-stationary-growth phase is dominated by factors involved in biodegradative metabolism, namely, a histidine ammonia lyase, three enzymes responsible for uptake and utilization of glycogen, the urease complex, and a subtilisin-like protease. Double-knockout mutants and complementation studies demonstrate inhibitory effects of MCPI and UhpC on the expression of a putative hemolysin transporter. The data partially delineate the spectrum of gene expression of Y. enterocolitica at environmental temperatures, providing evidence that an as-yet-unknown insect phase is part of the life cycle of this human pathogen.

Gene Expression Analysis of Corynebacterium glutamicum Subjected to Long-Term Lactic Acid Adaptation

Corynebacteria form an important part of the red smear cheese microbial surface consortium. To gain a better understanding of molecular adaptation due to low pH induced by lactose fermentation, the global gene expression profile of Corynebacterium glutamicum adapted to pH 5.7 with lactic acid under continuous growth in a chemostat was characterized by DNA microarray analysis. Expression of a total of 116 genes was increased and that of 90 genes was decreased compared to pH 7.5 without lactic acid, representing 7% of the genes in the genome. The up-regulated genes encode mainly transcriptional regulators, proteins responsible for export, import, and metabolism, and several proteins of unknown function. As much as 45% of the up-regulated open reading frames code for hypothetical proteins. These results were validated using real-time reverse transcription-PCR. To characterize the functions of 38 up-regulated genes, 36 single-crossover disruption mutants were generated and analyzed for their lactic acid sensitivities. However, only a sigB knockout mutant showed a highly significant negative effect on growth at low pH, suggesting a function in organic-acid adaptation. A sigE mutant already displayed growth retardation at neutral pH but grew better at acidic pH than the sigB mutant. The lack of acid-sensitive phenotypes in 34 out of 36 disrupted genes suggests either a considerable redundancy in acid adaptation response or coincidental effects. Other up-regulated genes included genes for ion transporters and metabolic pathways, including carbohydrate and respiratory metabolism. The enhanced expression of the nrd (ribonucleotide reductase) operon and a DNA ATPase repair protein implies a cellular response to combat acid-induced DNA damage. Surprisingly, multiple iron uptake systems (totaling 15% of the genes induced >or=2-fold) were induced at low pH. This induction was shown to be coincidental and could be attributed to iron-sequestering effects in complex media at low pH.

FTIR-based polyphasic identification of lactic acid bacteria isolated from traditional Greek Graviera cheese

This study used a combination of phenotypic, physical (Fourier Transformed Infra-Red [FTIR] spectroscopy) and molecular (RFLP and SSCP analysis of 16S rRNA genes) methods to identify the lactic acid bacteria (LAB) flora present in traditional Greek Graviera cheese after five weeks of ripening. A total of 300 isolates collected from high dilution plates of TSAYE (incubated at 30 °C), M-17 (22 °C) and M-17 (42 °C) agar media were clustered by FTIR and then representative strains of each cluster were cross-identified blindly by all methods. Based on their FTIR spectra, 282 isolates were LAB grouped in 28 clusters. The LAB species identified and their prevalence in the cheese samples were: Lactobacillus casei/paracasei (68.8%), Lactobacillus plantarum (19.5%), Streptococcus thermophilus (8.9%), Enterococcus faecium (2.1%), and Lactococcus lactis (0.7%). Also, Staphylococcus equorum (11 isolates), Corynebacterium sp. (5 isolates) and Brevibacterium sp. (1 isolate) were recovered from TSAYE. Comparative identification results showed that phenotypic and molecular methods were in mutual agreement as regards the LAB species identified. The present polyphasic identification approach based on rapid FTIR screening of 10-fold more isolates than a previous classical identification approach allowed or improved detection of few sub-dominant species; however the predominant LAB species in the cheese samples were the same with both approaches.

Comparison of strand-specific transcriptomes of enterohemorrhagic Escherichia coli O157:H7 EDL933 (EHEC) under eleven different environmental conditions including radish sprouts and cattle feces

BackgroundMultiple infection sources for enterohemorrhagic Escherichia coli O157:H7 (EHEC) are known, including animal products, fruit and vegetables. The ecology of this pathogen outside its human host is largely unknown and one third of its annotated genes are still hypothetical. To identify genetic determinants expressed under a variety of environmental factors, we applied strand-specific RNA-sequencing, comparing the SOLiD and Illumina systems.ResultsTranscriptomes of EHEC were sequenced under 11 different biotic and abiotic conditions: LB medium at pH4, pH7, pH9, or at 15°C; LB with nitrite or trimethoprim-sulfamethoxazole; LB-agar surface, M9 minimal medium, spinach leaf juice, surface of living radish sprouts, and cattle feces. Of 5379 annotated genes in strain EDL933 (genome and plasmid), a surprising minority of only 144 had null sequencing reads under all conditions. We therefore developed a statistical method to distinguish weakly transcribed genes from background transcription. We find that 96% of all genes and 91.5% of the hypothetical genes exhibit a significant transcriptional signal under at least one condition. Comparing SOLiD and Illumina systems, we find a high correlation between both approaches for fold-changes of the induced or repressed genes. The pathogenicity island LEE showed highest transcriptional activity in LB medium, minimal medium, and after treatment with antibiotics. Unique sets of genes, including many hypothetical genes, are highly up-regulated on radish sprouts, cattle feces, or in the presence of antibiotics. Furthermore, we observed induction of the shiga-toxin carrying phages by antibiotics and confirmed active biofilm related genes on radish sprouts, in cattle feces, and on agar plates.ConclusionsSince only a minority of genes (2.7%) were not active under any condition tested (null reads), we suggest that the assumption of significant genome over-annotations is wrong. Environmental transcriptomics uncovered hitherto unknown gene functions and unique regulatory patterns in EHEC. For instance, the environmental function of azoR had been elusive, but this gene is highly active on radish sprouts. Thus, NGS-transcriptomics is an appropriate technique to propose new roles of hypothetical genes and to guide future research.

Visual boosting in pixel-based visualizations

Pixel‐based visualizations have become popular, because they are capable of displaying large amounts of data and at the same time provide many details. However, pixel‐based visualizations are only effective if the data set is not sparse and the data distribution not random. Single pixels – no matter if they are in an empty area or in the middle of a large area of differently colored pixels – are perceptually difficult to discern and may therefore easily be missed. Furthermore, trends and interesting passages may be camouflaged in the sea of details. In this paper we compare different approaches for visual boosting in pixel‐based visualizations. Several boosting techniques such as halos, background coloring, distortion, and hatching are discussed and assessed with respect to their effectiveness in boosting single pixels, trends, and interesting passages. Application examples from three different domains (document analysis, genome analysis, and geospatial analysis) show the general applicability of the techniques and the derived guidelines.

Vibrio casei sp. nov., isolated from the surfaces of two French red smear soft cheeses

Three Gram-negative, rod-shaped, catalase- and oxidase-positive, facultatively anaerobic and motile bacteria, strains WS 4538, WS 4539T and WS 4540, were isolated from the surfaces of two fully ripened French red smear soft cheeses. Based on 16S rRNA gene sequence similarity, all three strains were shown to belong to the genus Vibrio. They are most closely related to Vibrio rumoiensis S-1T (96.3% similarity) and Vibrio litoralis MANO22DT (95.9%). DNA-DNA hybridization confirmed that all three isolates belong to the same species and clearly separated strain WS 4539T from V. rumoiensis DSM 19141T (38-42% relatedness) and V. litoralis DSM 17657(T) (28-37%). In contrast to their nearest relatives, the strains exhibited beta-galactosidase and aesculin hydrolase activities. A 14 bp insertion in the 16S rRNA gene sequence forms an elongated structure at helix 10 in the rRNA molecule and provides a tool for PCR-based identification of the novel species. Partial sequences of the housekeeping genes atpA, recA, rpoA and pyrH supported the conclusion that the three isolates constitute a separate species within the genus Vibrio. The name Vibrio casei sp. nov. is proposed for the novel taxon. Strain WS 4539T (=DSM 22364T =LMG 25240T; DNA G+C content 41.8 mol%) is the type strain and WS 4540 (=DSM 22378 =LMG 25241) is a reference strain.