A Burkholderia cenocepacia MurJ (MviN) homolog is essential for cell wall peptidoglycan synthesis and bacterial viability

Mohamed, Yasmine Fathy; Valvano, Miguel A.

doi:10.1093/glycob/cwu025

Cited by 25 publications

(33 citation statements)

References 54 publications

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“…A less commonly used but tightly controlled inducible promoter system is the rhamnose-inducible E. coli rhaSR-PrhaBAD (26,27). Although Valvano and coworkers showed that the E. coli rhaSR-PrhaBAD system is tightly controlled in Burkholderia cenocepacia (28) and used it to identify and study essential genes (29)(30)(31)(32), the functionality of this system in P. aeruginosa has not been reported. We constructed and tested two alternative inducible promoter systems for use in P. aeruginosa, miniTn7-lacI q -Ptac and miniTn7-rhaSR-PrhaBAD.…”

Section: Resultsmentioning

confidence: 99%

The Escherichia coli rhaSR-PrhaBAD Inducible Promoter System Allows Tightly Controlled Gene Expression over a Wide Range in Pseudomonas aeruginosa

Meisner

Goldberg

2016

Appl Environ Microbiol

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The araC-ParaBAD inducible promoter system is tightly controlled and allows gene expression to be modulated over a wide range in Escherichia coli, which has led to its widespread use in other bacteria. Although anecdotal evidence suggests that araCParaBAD is leaky in Pseudomonas aeruginosa, neither a thorough analysis of this inducible promoter system in P. aeruginosa nor a concerted effort to identify alternatives with improved functionality has been reported. Here, we evaluated the functionality of the araC-ParaBAD system in P. aeruginosa. Using transcriptional fusions to a lacZ reporter gene, we determined that the noninduced expression from araC-ParaBAD is high and cannot be reduced by carbon catabolite repression as it can in E. coli. Modulating translational initiation by altering ribosome-binding site strength reduced the noninduced activity but also decreased the maximal induced activity and narrowed the induction range. Integrating the inducible promoter system into the posttranscriptional regulatory network that controls catabolite repression in P. aeruginosa significantly decreased the noninduced activity and increased the induction range. In addition to these improvements in the functionality of the araC-ParaBAD system, we found that the lacI q -Ptac and rhaSR-PrhaBAD inducible promoter systems had significantly lower noninduced expression and were inducible over a broader range than araC-ParaBAD. We demonstrated that noninduced expression from the araC-ParaBAD system supported the function of genes involved in antibiotic resistance and tryptophan biosynthesis in P. aeruginosa, problems that were avoided with rhaSR-PrhaBAD. rhaSR-PrhaBAD is tightly controlled, allows gene expression over a wide range, and represents a significant improvement over araC-ParaBAD in P. aeruginosa. IMPORTANCEWe report the shortcomings of the commonly used Escherichia coli araC-ParaBAD inducible promoter system in Pseudomonas aeruginosa, successfully reengineered it to improve its functionality, and show that the E. coli rhaSR-PrhaBAD system is tightly controlled and allows inducible gene expression over a wide range in P. aeruginosa. Pseudomonas aeruginosa is a versatile Gram-negative bacterium that inhabits a variety of different environments. It is also an opportunistic human pathogen that causes acute infections in hospitalized patients as well as chronic infections in cystic fibrosis patients. Unfortunately, P. aeruginosa infections are becoming difficult to treat because of the increasing prevalence of multidrug (antibiotic) resistance (1). To improve the treatment of these infections, we need to understand which gene functions are essential for the growth of P. aeruginosa and develop new therapeutics to inhibit them. The study of essential genes is difficult because, by definition, inactivation of an essential gene is lethal to the cell. Analysis of essential genes generally involves the construction of conditional mutants, often accomplished by controlling the expression of a gene with an inducible promoter...

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Section: Resultsmentioning

confidence: 99%

The Escherichia coli rhaSR-PrhaBAD Inducible Promoter System Allows Tightly Controlled Gene Expression over a Wide Range in Pseudomonas aeruginosa

Meisner

Goldberg

2016

Appl Environ Microbiol

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“…Lastly, if the undecaprenyl moiety of lipid II stays in the hydrophobic core of the membrane during flipping, the flippase would have to have an open slot between two TMDs. The structural models of MurJ from E. coli (14) and B. cenocepacia (12) and of YtgP predict two of these features that are conserved in all three proteins. These open slots are present between TMDs 1 and 8 and TMDs 1 and 5 (see Fig.…”

Section: Discussionmentioning

confidence: 96%

“…The IM protein MurJ is essential for PG biogenesis and functions as the lipid II flippase (9)(10)(11). In agreement with this function, (i) MurJ depletion leads to the accumulation of PG precursors and cell lysis (10)(11)(12); (ii) MurJ belongs to the MVF family of the multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) exporter superfamily, which includes other flippases (13); (iii) MurJ contains a central hydrophilic cavity within the hydrophobic core of the membrane (14); (iv) residues within this hydrophilic cavity are essential for MurJ function (14); and (v) chemical inactivation of MurJ function rapidly stops lipid II flipping and leads to the accumulation of lipid II at the inner leaflet of the IM (9). Although we do not understand how MurJ functions in lipid II translocation, it likely shares functional features with members of the MOP exporter superfamily.…”

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confidence: 89%

“…Recent work has demonstrated that a MurJ homolog from the Gram-negative betaproteobacterium Burkholderia cenocepacia (MurJ BC ) is essential for PG biogenesis and predicted to be a structural homolog of E. coli MurJ (12). Furthermore, the two proteins are functionally interchangeable and share 54% identity (determined by Clustal Omega alignment [21]).…”

mentioning

confidence: 99%

“…Furthermore, the two proteins are functionally interchangeable and share 54% identity (determined by Clustal Omega alignment [21]). The essentiality of 5 charged residues that are homologous to essential residues located within the cavity of E. coli MurJ (14) has been investigated in MurJ BC ; of these residues, only 3 are essential for MurJ BC function in B. cenocepacia (12). Thus, evidence exists for both conserved and organism-specific requirements for MurJ activity.…”

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confidence: 99%

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Charge Requirements of Lipid II Flippase Activity in Escherichia coli

et al. 2014

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Peptidoglycan (PG) is an extracytoplasmic glycopeptide matrix essential for the integrity of the envelope of most bacteria. The PG building block is a disaccharide-pentapeptide that is synthesized as a lipid-linked precursor called lipid II. The translocation of the amphipathic lipid II across the cytoplasmic membrane is required for subsequent incorporation of the disaccharide-pentapeptide into PG. In Escherichia coli, the essential inner membrane protein MurJ is the lipid II flippase. Previous studies showed that 8 charged residues in the central cavity region of MurJ are crucial for function. Here, we completed the functional analysis of all 57 charged residues in MurJ and demonstrated that the respective positive or negative charge of the 8 aforementioned residues is required for proper MurJ function. Loss of the negative charge in one of these residues, D39, causes a severe defect in MurJ biogenesis; by engineering an intragenic suppressor mutation that restores MurJ biogenesis, we found that this charge is also essential for MurJ function. Because of the low level of homology between MurJ and putative orthologs from Gram-positive bacteria, we explored the conservation of these 8 charged residues in YtgP, a homolog from Streptococcus pyogenes. We found that only 3 positive charges are similarly positioned and essential in YtgP; YtgP possesses additional charged residues within its predicted cavity that are essential for function and conserved among Gram-positive bacteria. From these data, we hypothesize that some charged residues in the cavity region of MurJ homologs are required for interaction with lipid II and/or energy coupling during transport. Most bacteria produce a rigid peptidoglycan (PG) layer that is essential for defining cell shape and providing protection against osmotic lysis (1, 2). This mesh-like PG macromolecule consists of glycan strands of repeating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) disaccharide units that are bridged by the cross-linking of peptide stems extending from the MurNAc moiety. The PG layer is localized outside the cytoplasmic membrane, and as the cell grows, newly synthesized PG material must be incorporated into the preexisting polymer. Since the disaccharide-pentapeptide building blocks are made in the cytoplasm, the cytoplasmic membrane separates PG precursor synthesis from its utilization. Therefore, transport of the disaccharide-pentapeptide across the membrane is an essential step in PG biogenesis.In Escherichia coli, PG is present in the periplasm, the aqueous compartment between the inner and outer membranes (3). Like in all PG-producing bacteria, the disaccharide-pentapeptide PG precursor is synthesized onto the lipid carrier undecaprenol at the inner leaflet of the inner membrane (IM) (reviewed in references 4-7). This lipid-linked precursor, known as lipid II (undecaprenyl-pyrophosphoryl-MurNAc-[pentapeptide]-GlcNAc), must be flipped across the IM and delivered to the outer leaflet of the IM. Once lipid II is exposed to the periplasm, t...

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