Nutrient regime regulates complex transcriptional start site usage within a <i>Pseudoalteromonas</i> chitinase gene cluster

Delpin, Marina W; Goodman, Amanda E.

doi:10.1038/ismej.2009.54

Cited by 9 publications

(23 citation statements)

References 47 publications

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“…The overnight culture was diluted 1:100 into either 10 ml MMM GlcNAc, MMM NN GlcNAc, MMM maltose or MMM glt and grown with shaking. Cultures of OD 595 0.7-0.8 were treated with RNA protect (Qiagen, Venlo, Netherlands) and RNA was isolated using an RNeasy minikit (Qiagen) as described in detail by Delpin and Goodman (2009). Reverse transcription (RT)-PCR of S91 chiA was carried out as described in Delpin and Goodman (2009) using Thermoscript reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and the chiA gene-specific primer chiA-R895.…”

Section: Assay Of Chitinase Gene Expressionmentioning

confidence: 99%

“…Cultures of OD 595 0.7-0.8 were treated with RNA protect (Qiagen, Venlo, Netherlands) and RNA was isolated using an RNeasy minikit (Qiagen) as described in detail by Delpin and Goodman (2009). Reverse transcription (RT)-PCR of S91 chiA was carried out as described in Delpin and Goodman (2009) using Thermoscript reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and the chiA gene-specific primer chiA-R895. RNA was removed from transcribed cDNA by alkaline hydrolysis followed by glycogen precipitation, to recover the cDNA (Delpin and Goodman, 2009).…”

Section: Assay Of Chitinase Gene Expressionmentioning

confidence: 99%

“…Reverse transcription (RT)-PCR of S91 chiA was carried out as described in Delpin and Goodman (2009) using Thermoscript reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and the chiA gene-specific primer chiA-R895. RNA was removed from transcribed cDNA by alkaline hydrolysis followed by glycogen precipitation, to recover the cDNA (Delpin and Goodman, 2009). Successful cDNA synthesis was checked by PCR; the primer pair used was chiA-F776 and chiA-R895 (Delpin and Goodman, 2009).…”

Section: Assay Of Chitinase Gene Expressionmentioning

confidence: 99%

“…S91 chiA potentially has one s 70 -and two s 54 -dependent promoters, all of which are active during growth in MMM supplemented with glt with or without 0.1% (5 mM) GlcNAc (Delpin and Goodman, 2009). Many s 54 -dependent promoters have been found to be part of the nitrogen regulatory system (Zimmer et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

“…S91 is a chitinolytic marine bacterium (Techkarnjanaruk et al, 1997) in which the chitinase gene cluster, chiABC, is transcribed as an operon as well as separately from individual promoters possessed by each gene (Delpin and Goodman, 2009). In the transposon mutant strain S91CX, which has a lacZ reporter gene inserted under the control of the chiA promoter region, chiA promoter activity is regulated by growth conditions (Techkarnjanaruk et al, 1997).…”

Section: Introductionmentioning

confidence: 99%

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Nitrogen regulates chitinase gene expression in a marine bacterium

Delpin

Goodman

2009

The ISME Journal

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Ammonium concentration and nitrogen source regulate promoter activity and use for the transcription of chiA, the major chitinase gene of Pseudoalteromonas sp. S91 and S91CX, an S91 transposon lacZ fusion mutant. The activity of chiA was quantified by b-galactosidase assay of S91CX cultures containing different ammonium concentrations (NH 4 þ ; 0, 9.5 or 191 mM) and with different nitrogen sources (N-acetylglucosamine (GlcNAc) or glutamate (glt)). S91 chiA expression was found to depend on both the NH 4 þ concentration and source of nitrogen in marine minimal medium (MMM). Pseudoalteromonas sp. S91 and S91CX can use either GlcNAc or glt as a sole source of carbon in MMM containing a standard concentration of 9.5 mM NH 4 þ . Adding excess NH 4 þ , 20 times the standard concentration, to MMM significantly reduced chiA activity below that found in the presence of either GlcNAc or glt. When no NH 4 þ was added to MMM, S91CX was also able to use either GlcNAc or glt as a source of nitrogen; under these conditions chiA activity was significantly increased. Under all conditions tested, GlcNAc induced chiA activity significantly more than glt. Regulation of bacterial chitinases by nitrogen has not been previously reported. Transcriptional start point analysis of S91 chiA, using 5 0 RACE (ligation-anchored PCR), showed that during growth in MMM supplemented with (1) maltose (solely a carbon source for S91), chiA transcription occurred from only one putative r 70 -dependent promoter; (2) the chitin monomer GlcNAc, transcription initiated from two putative r 54 -dependent promoters and (3) glt, transcription initiated from all three putative promoters.

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Section: Assay Of Chitinase Gene Expressionmentioning

confidence: 99%

Section: Assay Of Chitinase Gene Expressionmentioning

confidence: 99%

Section: Assay Of Chitinase Gene Expressionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Nitrogen regulates chitinase gene expression in a marine bacterium

Delpin

Goodman

2009

The ISME Journal

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Mining of unexplored habitats for novel chitinases—chiA as a helper gene proxy in metagenomics

Cretoiu

Kielak

Al‐Soud

et al. 2012

Appl Microbiol Biotechnol

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The main objective of this study was to assess the abundance and diversity of chitin-degrading microbial communities in ten terrestrial and aquatic habitats in order to provide guidance to the subsequent exploration of such environments for novel chitinolytic enzymes. A combined protocol which encompassed (1) classical overall enzymatic assays, (2) chiA gene abundance measurement by qPCR, (3) chiA gene pyrosequencing, and (4) chiA gene-based PCR-DGGE was used. The chiA gene pyrosequencing is unprecedented, as it is the first massive parallel sequencing of this gene. The data obtained showed the existence across habitats of core bacterial communities responsible for chitin assimilation irrespective of ecosystem origin. Conversely, there were habitat-specific differences. In addition, a suite of sequences were obtained that are as yet unregistered in the chitinase database. In terms of chiA gene abundance and diversity, typical low-abundance/diversity versus high-abundance/diversity habitats was distinguished. From the combined data, we selected chitin-amended agricultural soil, the rhizosphere of the Arctic plant Oxyria digyna and the freshwater sponge Ephydatia fluviatilis as the most promising habitats for subsequent bioexploration. Thus, the screening strategy used is proposed as a guide for further metagenomics-based exploration of the selected habitats.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-012-4057-5) contains supplementary material, which is available to authorized users.

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A novel salt-tolerant chitobiosidase discovered by genetic screening of a metagenomic library derived from chitin-amended agricultural soil

Cretoiu

Berini

Kielak

et al. 2015

Appl Microbiol Biotechnol

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Here, we report on the construction of a metagenomic library from a chitin-amended disease-suppressive agricultural soil and its screening for genes that encode novel chitinolytic enzymes. The library, constructed in fosmids in an Escherichia coli host, comprised 145,000 clones containing inserts of sizes of 21 to 40 kb, yielding a total of approximately 5.8 GB of cloned soil DNA. Using genetic screenings by repeated PCR cycles aimed to detect gene sequences of the bacterial chitinase A-class (hereby named chi A genes), we identified and characterized five fosmids carrying candidate genes for chitinolytic enzymes. The analysis thus allowed access to the genomic (fosmid-borne) context of these genes. Using the chiA-targeted PCR, which is based on degenerate primers, the five fosmids all produced amplicons, of which the sequences were related to predicted chitinolytic enzyme-encoding genes of four different host organisms, including Stenotrophomonas maltophilia. Sequencing and de novo annotation of the fosmid inserts confirmed that each one of these carried one or more open reading frames that were predicted to encode enzymes active on chitin, including one for a chitin deacetylase. Moreover, the genetic contexts in which the putative chitinolytic enzyme-encoding genes were located were unique per fosmid. Specifically, inserts from organisms related to Burkholderia sp., Acidobacterium sp., Aeromonas veronii, and the chloroflexi Nitrolancetus hollandicus and/or Ktedonobacter racemifer were obtained. Remarkably, the S. maltophilia chiA-like gene was found to occur in two different genetic contexts (related to N. hollandicus/K. racemifer), indicating the historical occurrence of genetic reshufflings in this part of the soil microbiota. One fosmid containing the insert composed of DNA from the N. hollandicus-like organism (denoted 53D1) was selected for further work. Using subcloning procedures, its putative gene for a chitinolytic enzyme was successfully brought to expression in an E. coli host. On the basis of purified protein preparations, the produced protein was characterized as a chitobiosidase of 43.6 kDa, with a pI of 4.83. Given its activity spectrum, it can be typified as a halotolerant chitobiosidase.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-015-6639-5) contains supplementary material, which is available to authorized users.

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Nutrient regime regulates complex transcriptional start site usage within a Pseudoalteromonas chitinase gene cluster

Cited by 9 publications

References 47 publications

Nitrogen regulates chitinase gene expression in a marine bacterium

Nitrogen regulates chitinase gene expression in a marine bacterium

Mining of unexplored habitats for novel chitinases—chiA as a helper gene proxy in metagenomics

A novel salt-tolerant chitobiosidase discovered by genetic screening of a metagenomic library derived from chitin-amended agricultural soil

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