large-scale-screens-of-metagenomic-libraries

Large-Scale Screens of Metagenomic Libraries

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation.  Generating a ...

Myxococcus xanthus genetic mutants with characterized phenotypes were analysed for the ability to prey on susceptible bacteria. Quantification of predatory ability was scored by a newly developed method under conditions in which prey bacteria provided the only source of nutrients. These results were corroborated by data derived using a previously published protocol that measures predation in the presence of limited external nutrients. First, early developmental regulatory mutants were examined, because their likely functions in assessing the local nutrient status were predicted to be also important for predation. The results showed that predation efficiency is reduced by 64-80 % for mutants of three A-signalling components, AsgA, AsgC and AsgE, but not for AsgB. This suggests that an Asg regulon function that is separate from A-signal production is needed for predation. Besides the Asg components, mutations in the early developmental genes sdeK and csgA were also consistently observed to reduce predatory efficacy by 36 and 33 %, respectively. In contrast, later developmental components, such as DevRS, 4406 and PhoP4, did not appear to play significant roles in predation. The predatory abilities of mutants defective for motility were also tested. The data showed that adventurous, but not social, motility is required for predation in the assay. Also, mutants for components in the chemotaxis-like Frz system were found to be reduced in predation efficiency by between 62 and 85 %. In sum, it was demonstrated here that defects in development and development-related processes affect the ability of M. xanthus to prey on other bacteria.

Mutations affecting predation ability of the soil bacterium Myxococcus xanthus.

Mutationen, welche die Pr&#xE4;dation F&#xE4;higkeit des Bodenbakterium Myxococcus xanthus.

We report here the identification and characterization of a member of the Myxococcus xanthus SdeK signal transduction pathway, BrgE. This protein was identified as an SdeK-interacting component using a yeast two-hybrid screen, and we further confirmed this interaction by the glutathione S-transferase (GST) pulldown assay. Additional yeast two-hybrid analyses revealed that BrgE preferentially interacts with the putative amino-terminal sensor domain of SdeK, but not with the carboxy-terminal kinase domain. A brgE insertion strain was shown to be blocked in development between aggregation and mound formation, and decreased by 50-fold in viable spore production compared with the parental wild type. These phenotypes are similar to those of sdeK mutants. The brgE mutation also altered expression of a sample of Tn5 lac developmental markers that are also SdeK regulated. Finally, we demonstrated that a brgE sdeK double mutant has a more severe sporulation defect than either of the two single mutants, suggesting that BrgE and SdeK act synergistically to regulate wild-type levels of sporulation. In sum, these data suggest that BrgE operates as an auxiliary factor to stimulate the SdeK signal transduction pathway by directly binding to the amino-terminal sensor domain of SdeK.

BrgE is a regulator of Myxococcus xanthus development.

BrgE ist ein Regulator des xanthus Entwicklung.

An insertion in the rasA gene entirely blocked developmental aggregation and sporulation in Myxococcus xanthus while also reducing swarm expansion on a 0.3% agar surface. Data presented here demonstrate that rasA is required for extracellular fibril formation and social gliding motility.

RasA is required for Myxococcus xanthus development and social motility.

Eine Einf&uuml;gung des RasA-Gens blockiert vollst&auml;ndig Entwicklungsst&ouml;rungen Aggregation und Sporenbildung in Myxococcus Xanthus und gleichzeitig die Schwarm-Erweiterung auf einer 0,3 % Agar-Oberfl&auml;che reduzieren. Hier vorgestellte Daten zeigen, dass RasA f&uuml;r extrazellul&auml;re Fibrille Bildung und soziale gleiten Motilit&auml;t ist.

RasA ist f&uuml;r Myxococcus Xanthus-Entwicklung und soziale Beweglichkeit erforderlich.

Phosphate regulation is complex in the developmental prokaryote Myxococcus xanthus, and requires at least four two-component systems (TCSs). Here, the identification and characterization of a member of one TCS, designated PhoP4, is reported. phoP4 insertion and in-frame deletion strains caused spore viability to be decreased by nearly two orders of magnitude, and reduced all three development-specific phosphatase activities by 80-90 % under phosphate-limiting conditions. Microarray and quantitative PCR analyses demonstrated that PhoP4 is also required for appropriate expression of the predicted pstSCAB-phoU operon of inorganic phosphate assimilation genes. Unlike the case for the other three M. xanthus Pho TCSs, the chromosomal region around phoP4 does not contain a partner histidine kinase gene. Yeast two-hybrid analyses reveal that PhoP4 interacts reciprocally with PhoR2, the histidine kinase of the Pho2 TCS; however, the existence of certain phenotypic differences between phoP4 and phoR2 mutants suggests that PhoP4 interacts with another, as-yet unidentified, histidine kinase.

The response regulator PhoP4 is required for late developmental events in Myxococcus xanthus.

Phosphat-Verordnung ist in der Entwicklungsbiologie Prokaryoten Myxococcus Xanthus komplex und erfordert mindestens vier zwei-Komponenten-Systeme (TCSs). Hier wird die Identifizierung und Charakterisierung eines Mitglieds einer TCS, benannt PhoP4, berichtet. phoP4 einlegen und im Rahmen l&ouml;schen Belastungen verursacht Spore Lebensf&auml;higkeit um fast zwei Gr&ouml;&szlig;enordnungen vermindert werden und reduziert alle drei Entwicklung-spezifische Phosphatase-Aktivit&auml;ten um 80-90 % unter Phosphat einschr&auml;nkenden Bedingungen. Microarray und quantitative PCR-Analysen zeigten, dass PhoP4 auch f&uuml;r die entsprechenden Ausdruck von den vorhergesagten PstSCAB-PhoU-Operon von anorganischen Phosphat-Assimilation-Genen erforderlich ist. Anders als bei den anderen drei M. Xanthus Pho TCSs, die chromosomale Region um phoP4 enth&auml;lt kein Partner-Histidin-Kinase-gen. Hefe-zwei-Hybrid-Analysen zeigen, dass PhoP4 sich gegenseitig mit PhoR2, das Histidin-Kinase der TCS Pho2 interagiert; bestimmte ph&auml;notypische Unterschiede zwischen phoP4 und phoR2 Mutanten legt jedoch nahe, dass PhoP4 mit einem anderen, als interagiert-bisher nicht identifizierten, Histidin-Kinase.

Der Antwort-Regler PhoP4 ist f&uuml;r sp&auml;te Entwicklungsst&ouml;rungen Veranstaltungen in Myxococcus Xanthus erforderlich.

High-throughput identification of rRNA gene-containing clones in large insert metagenomic libraries is difficult, because of the high background of host ribosomal RNA (rRNA) and rRNA genes. To address this challenge, a membrane hybridization method was developed to identify all bacterial small subunit rRNA-containing fosmid clones of microbial community DNA from seven different depths in the North Pacific Subtropical Gyre. Out of 101,376 clones screened, 751 rDNA-containing clones were identified that grouped in approximately 60 different clades. Several rare sequences only remotely related to known groups were detected, including a Wolbachia-related sequence containing a putative intron or intervening sequence, as well as seven sequences from Order Myxococcales not previously detected in pelagic habitats. Stratified, depth-specific population structure was evident within both cultured and uncultured lineages. Conversely, some eurybathyal members of the genera Alcanivorax and Rhizobium shared identical small subunit ribosomal DNA sequences that were distributed from surface waters to the 4000 m depth. Comparison with similar analyses in Monterey Bay microbial communities revealed previously recognized, as well as some distinctive, depth-stratified partitioning that distinguished coastal from open ocean bacterioplankton populations. While some bias was evident in fosmid clone recovery in a few particular lineages, the overall phylogenetic group recovery and distributions were consistent with previous studies, as well as with direct shotgun sequence data from the same source DNA.

Phylogenetic analyses of ribosomal DNA-containing bacterioplankton genome fragments from a 4000 m vertical profile in the North Pacific Subtropical Gyre.

Hochdurchsatz-Identifizierung von rRNA-gen-haltigen Klone in gro&szlig;en Insert durchgef&uuml;hrte metagenomische Bibliotheken ist schwierig, wegen der hohen Hintergrund der Host Ribosomale RNA (rRNA) und rRNA gene. Um dieser Herausforderung zu begegnen, wurde eine Membran-Hybridisierung-Methode entwickelt, um alle bakteriellen kleine Untereinheit rRNA-haltigen Fosmid Klone der mikrobiellen Gemeinschaft DNA von sieben verschiedenen Tiefen in den subtropischen North Pacific Gyre zu identifizieren. Aus 101.376-Klone, geschirmt, 751 rDNA-haltigen Klone wurden identifiziert, die in ca. 60 verschiedenen Kladen gruppiert. Mehrere seltene Sequenzen, die nur aus der Ferne mit Bezug zu bekannten Gruppen gefunden, einschlie&szlig;lich einer Wolbachia-bezogene Sequenz enthalten ein mutma&szlig;liches Intron oder interveniert Sequenz sowie sieben Sequenzen aus Bestellung Myxococcales pelagische Lebensr&auml;ume nicht zuvor erkannt. Geschichtete, Tiefe-spezifische Bev&ouml;lkerungsstruktur zeigte sich innerhalb sowohl kultivierten und ungebildeten &Uuml;bertragungslinien. Umgekehrt teilten einige Eurybathyal Mitglieder der Gattungen Alcanivorax und Kn&ouml;llchenbakterien identische kleine Untereinheit ribosomale DNA-Sequenzen, die an die 4000 m Tiefe von Oberfl&auml;chengew&auml;ssern verteilt wurden. Vergleich mit &auml;hnlichen Analysen in Monterey Bay Mikrobengemeinschaften offenbart Tatbest&auml;nden, sowie einige markante, Tiefe geschichtet Partitionierung, die K&uuml;sten von offenen Ozean Bacterioplankton Populationen unterschieden. W&auml;hrend einige Vorurteile in Fosmid Klon Erholung in wenigen bestimmten &Uuml;bertragungslinien offensichtlich war, waren die insgesamt phylogenetische Gruppe Erholung und Distributionen konsistent mit fr&uuml;heren Studien sowie mit direkter Schrotflinte Sequenzdaten aus der gleichen Quelle DNA.

Phylogenetische Analysen der ribosomal DNA-haltigen Bacterioplankton Genom Fragmente aus einem 4000 m vertikalen Profil der subtropischen North Pacific Gyre.

The phylogenetic diversity of Bacteria and Archaea within a biodegraded, mesothermic petroleum reservoir in the Schrader Bluff Formation of Alaska was examined by two culture-independent methods based on fosmid and small-subunit rRNA gene PCR clone libraries. Despite the exclusion of certain groups by each method, there was overall no significant qualitative difference in the diversity of phylotypes recovered by the two methods. The resident Bacteria belonged to at least 14 phylum-level lineages, including the polyphyletic Firmicutes, which accounted for 36.2% of all small-subunit rRNA gene-containing (SSU(+)) fosmid clones identified. Members of uncultured divisions were also numerous and made up 35.2% of the SSU(+) fosmid clones. Clones from domain Archaea accounted for about half of all SSU(+) fosmids, suggesting that their cell numbers were comparable to those of the Bacteria in this microbial community. In contrast to the Bacteria, however, nearly all archaeal clones recovered by both methods were related to methanogens, especially acetoclastic methanogens, while the plurality of bacterial fosmid clones was affiliated with Synergistes-like acetogenic Firmicutes that possibly degrade longer-chain carboxylic acid components in the crude oil to acetate. These data suggest that acetate may be a key intermediary metabolite in this subsurface anaerobic food chain, which leads to methane production as the primary terminal electron sink.

Characterizing microbial diversity in production water from an Alaskan mesothermic petroleum reservoir with two independent molecular methods.

Die phylogenetische Vielfalt von Bakterien und Archaeen innerhalb einer Messung, Mesothermic Erd&ouml;l Stausee im Schrader Bluff Formation Alaska wurde von zwei kulturunabh&auml;ngig Methoden auf der Grundlage von Fosmid und klein-Untereinheit rRNA gen PCR Klon Bibliotheken untersucht. Trotz der Ausschluss bestimmter Gruppen jede Methode gab es insgesamt keine wesentlicher qualitativer Unterschied in der Verschiedenheit der Phylotypen wiederhergestellt, indem die beiden Methoden. Die ans&auml;ssigen Bakterien geh&ouml;rte mindestens 14 Stamm-Ebene-Linien, einschlie&szlig;lich der polyphyletic Firmicutes, was 36,2 % von ganz klein-Untereinheit rRNA-gen-haltigen (SSU(+))-Fosmid-Klone identifiziert. Mitglieder der ungebildeten Divisionen waren auch zahlreiche und 35,2 % der SSU(+) Fosmid Klone aus. Klone aus Dom&auml;ne Archaea entfielen rund die H&auml;lfte aller SSU(+) Fosmids, was darauf hindeutet, dass ihre Zell-Zahlen vergleichbar mit denen der Bakterien in dieser mikrobiellen Gemeinschaft waren. Im Gegensatz zu den Bakterien jedoch fast alle reitenden Klone wiederhergestellt, indem beide Methoden betrafen Methanogens, besonders Acetoclastic Methanogens, w&auml;hrend die Pluralit&auml;t der bakteriellen Fosmid Klone mit Synergistes-&auml;hnlichen Acetogenic Firmicutes angeschlossen wurde, die m&ouml;glicherweise l&auml;ngere Kette Carbons&auml;ure-Komponenten in das Roh&ouml;l Acetat abgebaut. Diese Daten legen nahe, dass Acetat ein wichtiger Vermittler Metabolit in diesem Untergrund anaeroben Nahrungskette, m&ouml;glicherweise Methan-Produktion als die prim&auml;re terminal Elektron-Senke f&uuml;hrt.

Charakterisierung mikrobieller Vielfalt in Produktion Wasser aus einem Alaskan Mesothermic Erd&ouml;l-Reservoir mit zwei unabh&auml;ngigen Molekulare Methoden.

To investigate the temporal, spatial and phylogenetic resolution of marine microbial community structure and variability, we designed and expanded a genome proxy array (an oligonucleotide microarray targeting marine microbial genome fragments and genomes), evaluated it against metagenomic sequencing, and applied it to time-series samples from the Monterey Bay. The expanded array targeted 268 microbial genotypes across much of the known diversity of cultured and uncultured marine microbes. The target abundances measured by the array were highly correlated to pyrosequence-based abundances (linear regression R(2) = 0.85-0.91, P < 0.0001). Fifty-seven samples from âˆ¼4 years in Monterey Bay were examined with the array, spanning the photic zone (0 m), the base of the surface mixed layer (30 m) and the subphotic zone (200 m). A significant portion of the expanded genome proxy array's targets showed signal (95 out of 268 targets present in â‰¥ 1 sample). The multi-year community survey showed the consistent presence of a core group of common and abundant targeted taxa at each depth in Monterey Bay, higher variability among shallow than deep samples, and episodic occurrences of more transient marine genotypes. The abundance of the most dominant genotypes peaked after strong episodic upwelling events. The genome-proxy array's ability to track populations of closely related genotypes indicated population shifts within several abundant target taxa, with specific populations in some cases clustering by depth or oceanographic season. Although 51 cultivated organisms were targeted (representing 19% of the array) the majority of targets detected and of total target signal (85% and âˆ¼92% respectively) were from uncultivated genotypes, often those derived from Monterey Bay. The array provided a relatively cost-effective approach (âˆ¼$15 per array) for surveying the natural history of uncultivated lineages.

Time-series analyses of Monterey Bay coastal microbial picoplankton using a 'genome proxy' microarray.

Um die zeitliche, r&auml;umliche und phylogenetische Aufl&ouml;sung der marine mikrobielle Gemeinschaftsstruktur und Variabilit&auml;t zu untersuchen, wir entwickelt und erweitert ein Genom Proxyarray (ein Oligonukleotid-Microarray targeting marine mikrobiellen Genoms Fragmente und Genome) es gegen durchgef&uuml;hrte metagenomische Sequenzierung ausgewertet und wendete sie auf Zeitreihen Proben von der Monterey Bay. Das erweiterte Array gezielt 268 mikrobielle Genotypen in weiten Teilen der bekannten Vielfalt der kultivierten und ungebildeten Meeresmikroben. Die Ziel-H&auml;ufigkeiten gemessen vom Array wurden auf Pyrosequence basierenden H&auml;ufigkeiten stark korreliert (lineare Regression Optionsbildschirm = 0,85-0.91, P &lt; 0,0001). Siebenundf&uuml;nfzig Proben aus &sim;4, die Jahre in Monterey Bay mit dem Array &uuml;ber die photic Zone (0 m), untersucht wurden gemischt die Basis der Oberfl&auml;che Schicht (30 m) und der subphotic Zone (200 m). Ein erheblicher Teil der erweiterten Genoms Proxyarray Ziele zeigte Signal (95 von 268 Ziele pr&auml;sentieren in &ge; 1 Probe). Die mehrj&auml;hrigen Community-Umfrage zeigte die konsequente Pr&auml;senz einer Kerngruppe Common und reichlich gezielte Taxa in jeder Tiefe in Monterey Bay, h&ouml;here Variabilit&auml;t unter flach als tiefe Samples und episodische vorkommen mehr vor&uuml;bergehenden marinen Genotypen. Die F&uuml;lle der dominierenden Genotypen erreichte Nachereignisse starke episodische Auftrieb. Das Genom-Proxy-Array F&auml;higkeit, Populationen von eng verfolgen bezogene Genotypen lie&szlig;en erkennen, dass die Bev&ouml;lkerung innerhalb mehrerer reichlich Ziel Taxa, mit bestimmten Populationen in einigen F&auml;llen clustering von Tiefe oder ozeanographische Saison wechselt. Obwohl 51 kultivierte Organismen gerichtet waren (f&uuml;r 19 % des Arrays) die Mehrheit der Ziele erkannt und der gesamten Ziel signalisieren (85 % und &sim;92 % beziehungsweise) von Wildpflanze Genotypen, oft diejenigen von Monterey Bay abgeleitet wurden. Das Array zur Verf&uuml;gung gestellt, eines relativ kosteng&uuml;nstigen Ansatzes (&sim;$ 15 pro Array) f&uuml;r die Vermessung der Naturgeschichte des unbebauten &Uuml;bertragungslinien.

Article	Year
Mutations affecting predation ability of the soil bacterium Myxococcus xanthus. Microbiology (Reading, England)\| PubMed ID: 15941994	2005
BrgE is a regulator of Myxococcus xanthus development. Molecular microbiology\| PubMed ID: 16045620	2005
RasA is required for Myxococcus xanthus development and social motility. Journal of bacteriology\| PubMed ID: 16166548	2005
The response regulator PhoP4 is required for late developmental events in Myxococcus xanthus. Microbiology (Reading, England)\| PubMed ID: 16735725	2006
Phylogenetic analyses of ribosomal DNA-containing bacterioplankton genome fragments from a 4000 m vertical profile in the North Pacific Subtropical Gyre. Environmental microbiology\| PubMed ID: 18494796	2008
Characterizing microbial diversity in production water from an Alaskan mesothermic petroleum reservoir with two independent molecular methods. Environmental microbiology\| PubMed ID: 18771500	2009
Time-series analyses of Monterey Bay coastal microbial picoplankton using a 'genome proxy' microarray. Environmental microbiology\| PubMed ID: 20695878	2011

Vinh D. Pham

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