Microbial Monitoring in the EDEN ISS Greenhouse, a Mobile Test Facility in Antarctica

The EDEN ISS greenhouse, integrated in two joined containers, is a confined mobile test facility in Antarctica for the development and optimization of new plant cultivation techniques for future space programs. The EDEN ISS greenhouse was used successfully from February to November 2018 for fresh food production for the overwintering crew at the Antarctic Neumayer III station. During the 9 months of operation, samples from the different plants, from the nutrition solution of the aeroponic planting system, and from diverse surfaces within the three different compartments of the container were taken [future exploration greenhouse (FEG), service section (SS), and cold porch (CP)]. Quantity as well as diversity of microorganisms was examined by cultivation. In case of the plant samples, microbial quantities were in a range from 102 to 104 colony forming units (CFU) per gram plant material. Compared to plants purchased from a German grocery, the produce hosted orders of magnitude more microorganisms than the EDEN ISS plants. The EDEN ISS plant samples contained mainly fungi and a few bacteria. No classical food associated pathogenic microorganism, like Escherichia and Salmonella, could be found. Probably due to the used cultivation approach, Archaea were not found in the samples. The bioburden in the nutrition solutions increased constantly over time but never reached critical values like 10² –10³ CFU per 100 mL in irrigation water as it is stated, e.g., for commercial European plant productions. The surface samples revealed high differences in the microbial burden between the greenhouse part of the container and the SS and CP part. However, the numbers of organisms (bacteria and fungi) found in the planted greenhouse were still not critical. The microbial loaded surfaces showed strong temporal as well as spatial fluctuations. In samples of the nutrition solution and the surface, the number of bacteria exceeded the amount of fungi by many times. For identification, 16S rRNA gene sequencing was performed for the isolated prokaryotic organisms. Phylogenetic analyses revealed that the most abundant bacterial phyla were Firmicutes and Actinobacteria. These phyla include plant- and human-associated bacterial species. In general, it could be shown that it is possible to produce edible fresh food in a remote environment and this food is safe for consumption from a microbiological point of view

The Two-Component Signal Transduction System CopRS of Corynebacterium glutamicum Is Required for Adaptation to Copper-Excess Stress

Copper is an essential cofactor for many enzymes but at high concentrations it is toxic for the cell. Copper ion concentrations ≥50 µM inhibited growth of Corynebacterium glutamicum. The transcriptional response to 20 µM Cu2+ was studied using DNA microarrays and revealed 20 genes that showed a ≥ 3-fold increased mRNA level, including cg3281-cg3289. Several genes in this genomic region code for proteins presumably involved in the adaption to copper-induced stress, e. g. a multicopper oxidase (CopO) and a copper-transport ATPase (CopB). In addition, this region includes the copRS genes (previously named cgtRS9) which encode a two-component signal transduction system composed of the histidine kinase CopS and the response regulator CopR. Deletion of the copRS genes increased the sensitivity of C. glutamicum towards copper ions, but not to other heavy metal ions. Using comparative transcriptome analysis of the ΔcopRS mutant and the wild type in combination with electrophoretic mobility shift assays and reporter gene studies the CopR regulon and the DNA-binding motif of CopR were identified. Evidence was obtained that CopR binds only to the intergenic region between cg3285 (copR) and cg3286 in the genome of C. glutamicum and activates expression of the divergently oriented gene clusters cg3285-cg3281 and cg3286-cg3289. Altogether, our data suggest that CopRS is the key regulatory system in C. glutamicum for the extracytoplasmic sensing of elevated copper ion concentrations and for induction of a set of genes capable of diminishing copper stress

The Complete Genome Sequence of Thermoproteus tenax: A Physiologically Versatile Member of the Crenarchaeota

Here, we report on the complete genome sequence of the hyperthermophilic Crenarchaeum Thermoproteus tenax (strain Kra 1, DSM 2078(T)) a type strain of the crenarchaeotal order Thermoproteales. Its circular 1.84-megabase genome harbors no extrachromosomal elements and 2,051 open reading frames are identified, covering 90.6% of the complete sequence, which represents a high coding density. Derived from the gene content, T. tenax is a representative member of the Crenarchaeota. The organism is strictly anaerobic and sulfur-dependent with optimal growth at 86 degrees C and pH 5.6. One particular feature is the great metabolic versatility, which is not accompanied by a distinct increase of genome size or information density as compared to other Crenarchaeota. T. tenax is able to grow chemolithoautotrophically (CO2/H-2) as well as chemoorganoheterotrophically in presence of various organic substrates. All pathways for synthesizing the 20 proteinogenic amino acids are present. In addition, two presumably complete gene sets for NADH:quinone oxidoreductase (complex I) were identified in the genome and there is evidence that either NADH or reduced ferredoxin might serve as electron donor. Beside the typical archaeal A(0)A(1)-ATP synthase, a membrane-bound pyrophosphatase is found, which might contribute to energy conservation. Surprisingly, all genes required for dissimilatory sulfate reduction are present, which is confirmed by growth experiments. Mentionable is furthermore, the presence of two proteins (ParA family ATPase, actin-like protein) that might be involved in cell division in Thermoproteales, where the ESCRT system is absent, and of genes involved in genetic competence (DprA, ComF) that is so far unique within Archaea

Identification, sequence analysis, and expression of a Corynebacterium glutamicum gene cluster encoding the three glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, and triosephosphate isomerase.

To investigate a possible chromosomal clustering of glycolytic enzyme genes in Corynebacterium glutamicum, a 6.4-kb DNA fragment located 5' adjacent to the structural phosphoenolpyruvate carboxylase (PEPCx) gene ppc was isolated. Sequence analysis of the ppc-proximal part of this fragment identified a cluster of three glycolytic genes, namely, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene gap, the 3-phosphoglycerate kinase (PGK) gene pgk, and the triosephosphate isomerase (TPI) gene tpi. The four genes are organized in the order gap-pgk-tpi-ppc and are separated by 215 bp (gap and pgk), 78 bp (pgk and tpi), and 185 bp (tpi and ppc). The predicted gene product of gap consists of 336 amino acids (M(r) of 36,204), that of pgk consists of 403 amino acids (M(r) of 42,654), and that of tpi consists of 259 amino acids (M(r) of 27,198). The amino acid sequences of the three enzymes show up to 62% (GAPDH), 48% (PGK), and 44% (TPI) identity in comparison with respective enzymes from other organisms. The gap, pgk, tpi, and ppc genes were cloned into the C. glutamicum-Escherichia coli shuttle vector pEK0 and introduced into C. glutamicum. Relative to the wild type, the recombinant strains showed up to 20-fold-higher specific activities of the respective enzymes. On the basis of codon usage analysis of gap, pgk, tpi, and previously sequenced genes from C. glutamicum, a codon preference profile for this organism which differs significantly from those of E. coli and Bacillus subtilis is presented

Identification of RamA, a Novel LuxR-type Transcriptional Regulator of Genes Involved in Acetate Metabolism of Corynebacterium glutamicum

In Corynebacterium glutamicum, the acetate-activating enzymes phosphotransacetylase and acetate kinase and the glyoxylate cycle enzymes isocitrate lyase and malate synthase are coordinately up-regulated in the presence of acetate in the growth medium. This regulation is due to transcriptional control of the respective pta-ack operon and the aceA and aceB genes, brought about at least partly by the action of the negative transcriptional regulator RamB. Using cell extracts of C. glutamicum and employing DNA affinity chromatography, mass spectrometry, and peptide mass fingerprinting, we identified a LuxR-type transcriptional regulator, designated RamA, which binds to the pta-ack and aceA/aceB promoter regions. Inactivation of the ramA gene in the genome of C. glutamicum resulted in mutant RG2. This mutant was unable to grow on acetate as the sole carbon and energy source and, in comparison to the wild type of C. glutamicum, showed very low specific activities of phosphotransacetylase, acetate kinase, isocitrate lyase, and malate synthase, irrespective of the presence of acetate in the medium. Comparative transcriptional cat fusion experiments revealed that this deregulation takes place at the level of transcription. By electrophoretic mobility shift analysis, purified His-tagged RamA protein was shown to bind specifically to the pta-ack and the aceA/aceB promoter regions, and deletion and mutation studies revealed in both regions two binding motifs each consisting of tandem A/C/TG4-6T/C or AC4-5A/G/T stretches separated by four or five arbitrary nucleotides. Our data indicate that RamA represents a novel LuxR-type transcriptional activator of genes involved in acetate metabolism of C. glutamicum