Analysis of the intestinal microbiota using SOLiD 16S rRNA gene sequencing and SOLiD shotgun sequencing

Background: Metagenomics seeks to understand microbial communities and assemblages by DNA sequencing. Technological advances in next generation sequencing technologies are fuelling a rapid growth in the number and scope of projects aiming to analyze complex microbial environments such as marine, soil or the gut. Recent improvements in longer read lengths and paired-sequencing allow better resolution in profiling microbial communities. While both 454 sequencing and Illumina sequencing have been used in numerous metagenomic studies, SOLiD sequencing is not commonly used in this area, as it is believed to be more suitable in the context of reference-guided projects. Results: To investigate the performance of SOLiD sequencing in a metagenomic context, we compared taxonomic profiles of SOLiD mate-pair sequencing reads with Sanger paired reads and 454 single reads. All sequences were obtained from the bacterial 16S rRNA gene, which was amplified from microbial DNA extracted from a human fecal sample. Additionally, from the same fecal sample, complete genomic microbial DNA was extracted and shotgun sequenced using SOLiD sequencing to study the composition of the intestinal microbiota and the existing microbial metabolism. We found that the microbiota composition of 16S rRNA gene sequences obtained using Sanger, 454 and SOLiD sequencing provide results comparable to the result based on shotgun sequencing. Moreover, with SOLiD sequences we obtained more resolution down to the species level. In addition, the shotgun data allowed us to determine a functional profile using the databases SEED and KEGG. Conclusions: This study shows that SOLiD mate-pair sequencing is a viable and cost-efficient option for analyzing a complex microbiome. To the best of our knowledge, this is the first time that SOLiD sequencing has been used in a human sample. Keywords: Metagenomics; Intestinal Microbiota; Next-Generation Sequencing; SOLiD Mate-Pair Sequencing; Human Fecal SamplePublished versio

Transcriptional Response of Mucoid Pseudomonas aeruginosa to Human Respiratory Mucus

Adaptation of bacterial pathogens to a host can lead to the selection and accumulation of specific mutations in their genomes with profound effects on the overall physiology and virulence of the organisms. The opportunistic pathogen Pseudomonas aeruginosa is capable of colonizing the respiratory tract of individuals with cystic fibrosis (CF), where it undergoes evolution to optimize survival as a persistent chronic human colonizer. The transcriptome of a host-adapted, alginate-overproducing isolate from a CF patient was determined following growth of the bacteria in the presence of human respiratory mucus. This stable mucoid strain responded to a number of regulatory inputs from the mucus, resulting in an unexpected repression of alginate production. Mucus in the medium also induced the production of catalases and additional peroxide-detoxifying enzymes and caused reorganization of pathways of energy generation. A specific antibacterial type VI secretion system was also induced in mucus-grown cells. Finally, a group of small regulatory RNAs was identified and a fraction of these were mucus regulated. This report provides a snapshot of responses in a pathogen adapted to a human host through assimilation of regulatory signals from tissues, optimizing its long-term survival potential

The citronellol metabolism in Pseudomonads - functional assignment of involved genes and their products

Citronellol und Geraniol sind in der Natur als Duftstoffe weit verbreitete azyklische Terpenoide, die aufgrund ihrer β-ständigen Methylgruppe nicht über β-Oxidation abgebaut werden können. In jüngster Zeit konnten zwei in den Citronellolabbau involvierte Gencluster in P. aeruginosa PAO1 identifiziert werden, das atu (acyclic terpene utilisation)- und das liu (leucine/isovalerate utilisation)-Gencluster (FÖRSTER-FROMME ET AL., 2006, HÖSCHLE ET AL., 2005). Bisher konnten nur die Gene der in den Citronellolstoffwechsel involvierten Carboxylasen funktionell identifiziert werden (HÖSCHLE ET AL., 2005). In dieser Arbeit wurden weitere Gene der Cluster funktionell zugeordnet. - In P. citronellolis wurde ein Gencluster (atuABCDEFGH) identifiziert und sequenziert, dessen Gene sehr hohe Identitäten zu den atu-Genen von P. aeruginosa PAO1 aufwiesen. Dieses Gencluster wurde als atu-Gencluster von P. citronellolis annotiert. Eine Insertionmutante in atuA zeigte, daß dieses Gen essentiell für den Citronellolabbau ist. - In P. citronellolis konnte anhand zweier Transposonmutanten in mqoB gezeigt werden, daß die Malat-Quinon-Oxidoreduktase (MqoB) eine essentielle Rolle beim Abbau von Terpenoiden spielt, da die Endprodukte des Citronellolabbaus in den Glyoxylatstoffwechsel eingehen und die Malat-Quinon-Oxidoreduktase ein essentielles Enzym dieses Stoffwechselweges ist. - In P. aeruginosa PAO1 konnte die Funktion der Citronellyl-CoA-Dehydrogenase AtuD zugeordnet werden. AtuD wurde exprimiert und gereinigt und eine spezifische Aktivität von 850 ± 37 mU/mg für den Umsatz von Citronellyl-CoA ermittelt. Der KM-Wert für Citronellyl-CoA beträgt 1,6 ± 0,3 µM. Das Reaktionsprodukt Geranyl-CoA wurde mit HPLC und Massenspektrometrie nachgewiesen. AtuD ist hochspezifisch für Citronellyl-CoA, da Isovaleryl-CoA oder Octanoyl-CoA nicht umsetzt werden. Durch 2D-Gelanalysen wurde eine weitere Acyl-CoA-Dehydrogenase als spezifisch beim Wachstum auf Terpenoiden erkannt und als PA1535-Genprodukt identifiziert. Das PA1535-Genprodukt wurde heterolog in E. coli exprimiert, gereinigt und die Substratspezifität bestimmt. Das Enzym setzte Citronellyl-CoA mit 2450 ± 26 mU/mg (KM-Wert 18 ± 1,1 µM) und Octanoyl-CoA mit 610 ± 10 mU/mg (KM-Wert 130 ± 9,8 µM) um. Isovaleryl-CoA stellt kein geeignetes Substrat dar. - In P. aeruginosa PAO1 wurde die Funktion der Isovaleryl-CoA-Dehydrogenase LiuA zugeordnet. Gereinigtes LiuA setzte Isovaleryl-CoA mit 1620 ± 55 mU/mg (KM-Wert 2,3 ± 0,4 µM) um. Langkettige CoA-Thioester wie Citronellyl-CoA oder Octanoyl-CoA wurden nicht umgesetzt. - In P. aeruginosa PAO1 wurde das stromaufwärts des atu-Genclusters liegende Gen (PA2885) durch Insertionsmutangenese als Repressor des atu-Genclusters identifiziert (atuR). AtuR gehört zur TetR-Familie. In dem intergenischen Bereich zwischen atuR und atuA (280 bp) wurde eine Region mit Ähnlichkeit zu σ-70 Promotoren sowie partiell überlappend zwei perfekte inverted repeats gefunden. Heterolog exprimiertes und gereinigtes AtuRHis6 war in der Lage, in Gelshift-Experimenten spezifisch an den intergenischen Bereich zu binden. Für eine vollständige Bindung waren beide inverted repeats, nicht aber die -10- und die -35-Region notwendig. Citronellol, Geraniol, Citronellal, Geranial, Citronellsäure und Geranylsäure, nicht aber Leucin, Isovaleriansäure, 3-Methylcrotonsäure, Phytol und Eukalyptol verhinderten die Bindung von AtuR und stellen somit offenbar Effektoren von AtuR dar. - Mithilfe von Proteomanalysen wurde gezeigt, daß sechs von acht Atu-Proteinen und vier von fünf Liu-Proteinen spezifisch beim Wachstum auf azyklischen Terpenen, bzw. Isovalerat gebildet werden. Des weiteren wurden 16 weitere Proteine identifiziert, die vermutlich ebenfalls am Citronellolstoffwechsel beteiligt sind. - Bei P. aeruginosa POA1 wurden alle Gene des atu-Genclusters durch Insertion von pKnockout-G (WINDGASSEN ET AL., 2000) inaktiviert. Es wurde gezeigt, daß fünf der atu-Gene (atuA, atuB, atuC, atuD, atuF) essentiell für den Citronellolabbau waren. Insertionsmutanten in atuE, atuG und atuH zeigten keinen Phänotyp.Citronellol and geraniol are widespread scents with acyclic terpenoid molecule structures in nature. Because of their β-methyl-branched carbon skeleton they can not be degraded via β-oxidation. Recently two gene clusters involved in the citronellol degradation pathway could be identified in P. aeruginosa PAO1, the atu (acyclic terpene utilisation)- and the liu (leucine/isolvalerate utilisation)-gene cluster (FÖRSTER-FROMME ET AL., 2006, HÖSCHLE ET AL., 2005). Up to now only genes coding the carboxylases involved in the citronellol degradation pathway could be identified (HÖSCHLE ET AL., 2005). In the present work several other genes of the gene clusters were functionally assigned. - A gene cluster (atuRABCDEFGH) was identified and sequenced in P. citronellolis; the genes showed very high similarities to the atu-genes of P. aeruginosa PAO1. An insertion mutant in atuA was unable to utilise acyclic terpenes and indicated that this gene is essential for citronellol degradation. - Investigation of two P. citronellolis transposon mutants in mqoB showed that the malate:quinone oxidoreductase (MQO) plays an essential role in the degradation of terpenoids. The end products of the citronellol degradation pathway enter the glyoxylate cycle and MQO is an essential enzyme of this pathway. - In P. aeruginosa PAO1 the function of the citronellyl-CoA-dehydrogenase was assigned to AtuD. AtuD was expressed, purified and a specific activity of 850 ± 37 mU/mg for citronellyl-CoA was determined. The KM value for citronellyl-CoA is 1,6 ± 0,3 µM. The reaction product geranyl-CoA was identified by mass spectrometry. AtuD is highly specific for citronellyl-CoA while it is inactive with isovaleryl-CoA and octanoyl-CoA. 2D-gel-analysis showed that an additional acyl-CoA-dehydrogenase was expressed specifically during growth on terpenoids and it was identified as PA1535 gene product. The PA1535 gene product was expressed in E. coli, purified and its substrate specifity was determined. The enzyme converted citronellyl-CoA with 2450 ± 26 mU/mg (KM value18 ± 1,1 µM) and octanoyl-CoA with 610 ± 10 mU/mg (KM value130 ± 9,8 µM). The enzyme was inactive with isovaleryl-CoA. - The function of the isovaleryl-CoA-dehydrogenase was assigned to LiuA in P. aeruginosa PAO1. LiuA was expressed and converts isovaleryl-CoA with 1620 ± 55 mU/mg (KM value 2,3 ± 0,4 µM). LiuA was inactive with long chain acyl-CoA thioesters like citronellyl-CoA or octanoyl-CoA. - The gene upstream of the atu-gene cluster (PA2885) was identified as the repressor of the atu-gene cluster (AtuR) in P. aeruginosa PAO1. AtuR is a member of the TetR family. An area with similarities to σ-70 promoters and partial overlapping two perfect inverted repeats was identified in the intergenic region between atuR and atuA (280 bp). Heterologically expressed and purified AtuRHis6 was able to bind specifically to the intergenic region in gel shift experiments. Both inverted repeats but not the -10 and the -35-region were necessary for complete binding. Citronellol, geraniol, citronellal, geranial, citronellate and geranylate but not leucine, isovaleric acid, 3-methylcrotonic acid, phytol and eucalyptol prevented AtuR from binding and apparently represent the effectors of AtuR. - Proteom analysis showed that six of eight Atu-proteins and four of five Liu-proteins were specifically expressed during growth on acyclic terpenes and isovalerate, respectively. Furthermore 16 additional proteins were identified which are supposed to be involved in the citronellol degradation pathway. - All atu-genes of the atu-gene cluster were inactivated by insertion of pKnockout-G (WINDGASSEN ET AL., 2000) into the genes in P. aeruginosa PAO1. Five of them (atuA, atuB, atuC, atuD, atuF) turned out to be essential for citronellol utilisation. Insertion in atuE, atuG and atuH showed no phenotype

Identification of Genes and Proteins Necessary for Catabolism of Acyclic Terpenes and Leucine/Isovalerate in Pseudomonas aeruginosa

Geranyl-coenzyme A (CoA)-carboxylase (GCase; AtuC/AtuF) and methylcrotonyl-CoA-carboxylase (MCase; LiuB/LiuD) are characteristic enzymes of the catabolic pathway of acyclic terpenes (citronellol and geraniol) and of saturated methyl-branched compounds, such as leucine or isovalerate, respectively. Proteins encoded by two gene clusters (atuABCDEFGH and liuRABCDE) of Pseudomonas aeruginosa PAO1 were essential for acyclic terpene utilization (Atu) and for leucine and isovalerate utilization (Liu), respectively, as revealed by phenotype analysis of 10 insertion mutants, two-dimensional gel electrophoresis, determination of GCase and MCase activities, and Western blot analysis of wild-type and mutant strains. Analysis of the genome sequences of other pseudomonads (P. putida KT2440 and P. fluorescens Pf-5) revealed candidate genes for Liu proteins for both species and candidate genes for Atu proteins in P. fluorescens. This result concurred with the finding that P. fluorescens, but not P. putida, could grow on acyclic terpenes (citronellol and citronellate), while both species were able to utilize leucine and isovalerate. A regulatory gene, atuR, was identified upstream of atuABCDEFGH and negatively regulated expression of the atu gene cluster