Characterization of the promoter and upstream activating sequence from the Pseudomonas alcaligenes lipase gene

Cox, Manon; Gerritse, Gijs; Dankmeyer, Lydia; Quax, Wim J.

doi:10.1016/s0168-1656(00)00397-7

Cited by 16 publications

(14 citation statements)

References 28 publications

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“…The transcription start of the lipase operon in P. alcaligenes is similar to the consensus of 54-type promoters with a UAS (7). This points to a mechanism of positive control of lipase gene transcription by a regulatory protein similar to the one found for other 54 promoters (30).…”

Section: Discussionsupporting

confidence: 53%

“…Upon the binding of the regulatory protein to this UAS and to 54 RNA polymerase, the expression of the gene is induced. Within the upstream region of the lipase promoter region of P. alcaligenes, a sequence that is homologous to the Nif type of UASs (consensus, TGT-N10-ACA [3,25]) was identified (7). Mutational analysis of this UAS confirmed an involvement in lipase regulation (7).…”

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confidence: 90%

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Lipase Expression in Pseudomonas alcaligenes Is Under the Control of a Two-Component Regulatory System

Krzeslak

Gerritse²,

Merkerk

et al. 2008

Appl Environ Microbiol

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Preliminary observations in a large-scale fermentation process suggested that the lipase expression of Pseudomonas alcaligenes can be switched on by the addition of certain medium components, such as soybean oil. In an attempt to elucidate the mechanism of induction of lipase expression, we have set up a search method for genes controlling lipase expression by use of a cosmid library containing fragments of P. alcaligenes genomic DNA. A screen for lipase hyperproduction resulted in the selection of multiple transformants, of which the best-producing strains comprised cosmids that shared an overlapping genomic fragment. Within this fragment, two previously unidentified genes were found and named lipQ and lipR. Their encoded proteins belong to the NtrBC family of regulators that regulate gene expression via binding to a specific upstream activator sequence (UAS). Such an NtrC-like UAS was identified in a previous study in the P. alcaligenes lipase promoter, strongly suggesting that LipR acts as a positive regulator of lipase expression. The regulating role could be confirmed by down-regulated lipase expression in a strain with an inactivated lipR gene and a threefold increase in lipase yield in a large-scale fermentation when expressing the lipQR operon from the multicopy plasmid pLAFR3. Finally, cell extracts of a LipR-overexpressing strain caused a retardation of the lipase promoter fragment in a band shift assay. Our results indicate that lipase expression in Pseudomonas alcaligenes is under the control of the LipQR two-component system.

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Section: Discussionsupporting

confidence: 53%

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confidence: 90%

Lipase Expression in Pseudomonas alcaligenes Is Under the Control of a Two-Component Regulatory System

Krzeslak

Gerritse²,

Merkerk

et al. 2008

Appl Environ Microbiol

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“…S2 in the supplemental material). Mutational analysis of the lipA promoter of P. alcaligenes (7) and preliminary data on the hutU promoter of P. aeruginosa (16) reveal similar conserved palindromic sequences. Alignment of their sequences leads us to propose a consensus CbrB binding site, in which the spacer consists of 3 or 12 variable nucleotides (designated N 3 and N 12 , respectively).…”

Section: Vol 193 2011mentioning

confidence: 93%

“…The CbrB protein is a transcriptional activator for 54 RNA polymerase and belongs to the NtrC family of response regulators (30). Genetic evidence indicates that CbrB positively controls the expression of the crcZ sRNA gene in P. aeruginosa (36), the lipA (lipase) gene in Pseudomonas alcaligenes (7,18), and the hutU (histidine utilization) operon of P. aeruginosa and P. fluorescens (16,40). The fact that cbrB and crcZ mutants of P. aeruginosa have similar, but not identical, phenotypes suggests that most, but not all, activities of the CbrA/CbrB system are mediated by the sRNA CrcZ (36).…”

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Promoter Recognition and Activation by the Global Response Regulator CbrB in Pseudomonas aeruginosa

et al. 2011

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In Pseudomonas aeruginosa, the CbrA/CbrB two-component system is instrumental in the maintenance of the carbon-nitrogen balance and for growth on carbon sources that are energetically less favorable than the preferred dicarboxylate substrates. The CbrA/CbrB system drives the expression of the small RNA CrcZ, which antagonizes the repressing effects of the catabolite repression control protein Crc, an RNA-binding protein.Dicarboxylates appear to cause carbon catabolite repression by inhibiting the activity of the CbrA/CbrB system, resulting in reduced crcZ expression. Here we have identified a conserved palindromic nucleotide sequence that is present in upstream activating sequences (UASs) of promoters under positive control by CbrB and 54 RNA polymerase, especially in the UAS of the crcZ promoter. Evidence for recognition of this palindromic sequence by CbrB was obtained in vivo from mutational analysis of the crcZ promoter and in vitro from electrophoretic mobility shift assays using crcZ promoter fragments and purified CbrB protein truncated at the N terminus. Integration host factor (IHF) was required for crcZ expression. CbrB also activated the lipA (lipase) promoter, albeit less effectively, apparently by interacting with a similar but less conserved palindromic sequence in the UAS of lipA. As expected, succinate caused CbrB-dependent catabolite repression of the lipA promoter. Based on these results and previously published data, a consensus CbrB recognition sequence is proposed. This sequence has similarity to the consensus NtrC recognition sequence, which is relevant for nitrogen control.Pseudomonas aeruginosa, like other fluorescent pseudomonads, is a metabolically versatile bacterium; it utilizes more than 100 different organic substrates for growth (37). This versatility requires a complex regulatory network that ensures the cellular carbon-nitrogen balance and determines the order in which growth substrates are degraded. In general, substrates that yield high energy and promote fast growth are degraded preferentially. For instance, intermediates of the tricarboxylic acid (TCA) cycle such as succinate prevent glucose degradation in P. aeruginosa (21). As a result, growth on a mixture of succinate and glucose is biphasic (diauxic) because the bacterium utilizes first succinate and then glucose (39). The underlying mechanism of carbon catabolite repression involves the CbrA/ CbrB two-component system (16,30), the small RNA (sRNA) CrcZ (36), and the RNA-binding protein Crc (6,22,25,26,33) as key regulatory elements. They are conserved in fluorescent pseudomonads (38; Pseudomonas Genome Database).

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“…Acidogens, such as Bacillus, Alcaligenes, and Pseudomonas spp., also are associated with these lipolytic activities, but whether these organisms form part of the primary lipolytic bacterial group is unknown (Dartois et al, 1994;Wilhelm et al, 1999;Cox et al, 2001). The presence of photosynthetic bacteria in anaerobic digesters or fermentors has not been reported.…”

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Reuse of Organic Matter from Coagulated Sludge Using a Membrane-Coupled Anaerobic Organic Acid Fermentor in Advanced Municipal Wastewater Treatment

Kim¹,

Chung²

2012

Environmental Engineering Science

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This study focused on the development of an advanced municipal wastewater treatment process using a membrane-coupled anaerobic organic acid fermentor (MAOF) to remove dissolved organic matter from coagulated sludge. Intermittent ozone bubbling was effective in preventing increases in permeation resistance that were caused by particle accumulation on the membrane surface and in maintaining a high permeation flux. With regard to taxonomy, approximately 20 isolates were identified in the MAOF and are believed to represent the principal cell types in the fermentor. MAOF, coupled with intermittent ozone bubbling, is an effective system that can be implemented to recover organic matter that is used in biological denitrification or other basic materials.

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Characterization of the promoter and upstream activating sequence from the Pseudomonas alcaligenes lipase gene

Cited by 16 publications

References 28 publications

Lipase Expression in Pseudomonas alcaligenes Is Under the Control of a Two-Component Regulatory System

Lipase Expression in Pseudomonas alcaligenes Is Under the Control of a Two-Component Regulatory System

Promoter Recognition and Activation by the Global Response Regulator CbrB in Pseudomonas aeruginosa

Reuse of Organic Matter from Coagulated Sludge Using a Membrane-Coupled Anaerobic Organic Acid Fermentor in Advanced Municipal Wastewater Treatment

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