scholarly journals Mechanical Genomic Studies Reveal the Role of D-Alanine Metabolism inPseudomonas aeruginosaCell Stiffness

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Rishi R. Trivedi ◽  
John A. Crooks ◽  
George K. Auer ◽  
Joel Pendry ◽  
Ilona P. Foik ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Pseudomonas Aeruginosa ◽  
Cell Wall ◽  
High Throughput ◽  
Bacterial Cell ◽  
Mass Spectrometry Analysis ◽  
Cell Stiffness ◽  
Cross Linking ◽  
Content Type ◽  
In Cells

ABSTRACTThe stiffness of bacteria prevents cells from bursting due to the large osmotic pressure across the cell wall. Many successful antibiotic chemotherapies target elements that alter mechanical properties of bacteria, and yet a global view of the biochemistry underlying the regulation of bacterial cell stiffness is still emerging. This connection is particularly interesting in opportunistic human pathogens such asPseudomonas aeruginosathat have a large (80%) proportion of genes of unknown function and low susceptibility to different families of antibiotics, including beta-lactams, aminoglycosides, and quinolones. We used a high-throughput technique to study a library of 5,790 loss-of-function mutants covering ~80% of the nonessential genes and correlatedP. aeruginosaindividual genes with cell stiffness. We identified 42 genes coding for proteins with diverse functions that, when deleted individually, decreased cell stiffness by >20%. This approach enabled us to construct a “mechanical genome” forP. aeruginosa.d-Alanine dehydrogenase (DadA) is an enzyme that convertsd-Ala to pyruvate that was included among the hits; when DadA was deleted, cell stiffness decreased by 18% (using multiple assays to measure mechanics). An increase in the concentration ofd-Ala in cells downregulated the expression of genes in peptidoglycan (PG) biosynthesis, including the peptidoglycan-cross-linking transpeptidase genesponAanddacC. Consistent with this observation, ultraperformance liquid chromatography-mass spectrometry analysis of murein fromP. aeruginosacells revealed thatdadAdeletion mutants contained PG with reduced cross-linking and altered composition compared to wild-type cells.IMPORTANCEThe mechanical properties of bacteria are important for protecting cells against physical stress. The cell wall is the best-characterized cellular element contributing to bacterial cell mechanics; however, the biochemistry underlying its regulation and assembly is still not completely understood. Using a unique high-throughput biophysical assay, we identified genes coding proteins that modulate cell stiffness in the opportunistic human pathogenPseudomonas aeruginosa. This approach enabled us to discover proteins with roles in a diverse range of biochemical pathways that influence the stiffness ofP. aeruginosacells. We demonstrate thatd-Ala—a component of the peptidoglycan—is tightly regulated in cells and that its accumulation reduces expression of machinery that cross-links this material and decreases cell stiffness. This research demonstrates that there is much to learn about mechanical regulation in bacteria, and these studies revealed new nonessentialP. aeruginosatargets that may enhance antibacterial chemotherapies or lead to new approaches.

scholarly journals Novel Quorum-Sensing Peptides Mediating Interspecies Bacterial Cell Death

mBio ◽  
2013 ◽  
Vol 4 (3) ◽  
Author(s):  
Sathish Kumar ◽  
Ilana Kolodkin-Gal ◽  
Hanna Engelberg-Kulka
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Death ◽  
Bacillus Subtilis ◽  
Quorum Sensing ◽  
Bacterial Cell ◽  
Content Type ◽  
E Coli ◽  
New Class ◽  
Bacterial Cell Death ◽  
Induced Module

ABSTRACTEscherichia colimazEFis a toxin-antitoxin stress-induced module mediating cell death. It requires the quorum-sensing signal (QS) “extracellular death factor” (EDF), the penta-peptide NNWNN (EcEDF), enhancing the endoribonucleolytic activity ofE. colitoxin MazF. Here we discovered thatE. coli mazEF-mediated cell death could be triggered by QS peptides from the supernatants (SN) of the Gram-positive bacteriumBacillus subtilisand the Gram-negative bacteriumPseudomonas aeruginosa. In the SN ofB. subtilis, we found one EDF, the hexapeptide RGQQNE, calledBsEDF. In the SN ofP. aeruginosa, we found three EDFs: the nonapeptide INEQTVVTK, calledPaEDF-1, and two hexadecapeptides, VEVSDDGSGGNTSLSQ, calledPaEDF-2, and APKLSDGAAAGYVTKA, calledPaEDF-3. When added to a dilutedE. colicultures, each of these peptides acted as an interspecies EDF that triggeredmazEF-mediated death. Furthermore, though their sequences are very different, each of these EDFs amplified the endoribonucleolytic activity ofE. coliMazF, probably by interacting with different sites onE. coliMazF. Finally, we suggest that EDFs may become the basis for a new class of antibiotics that trigger death from outside the bacterial cells.IMPORTANCEBacteria communicate with one another via quorum-sensing signal (QS) molecules. QS provides a mechanism for bacteria to monitor each other’s presence and to modulate gene expression in response to population density. Previously, we addedE. coliEDF (EcEDF), the peptide NNWNN, to this list of QS molecules. Here we extended the group of QS peptides to several additional different peptides. The new EDFs are produced by two other bacteria,Bacillus subtilisandPseudomonas aeruginosa. Thus, in this study we established a “new family of EDFs.” This family provides the first example of quorum-sensing molecules participating in interspecies bacterial cell death. Furthermore, each of these peptides provides the basis of a new class of antibiotics triggering death by acting from outside the cell.

scholarly journals Alginate Oligosaccharide-Induced Modification of the lasI-lasR and rhlI-rhlR Quorum-Sensing Systems in Pseudomonas aeruginosa

2018 ◽  
Vol 62 (5) ◽  
Author(s):  
Alison A. Jack ◽  
Saira Khan ◽  
Lydia C. Powell ◽  
Manon F. Pritchard ◽  
Konrad Beck ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Laser Scanning ◽  
Homoserine Lactone ◽  
Laser Scanning Microscopy ◽  
Extracellular Dna ◽  
Mass Spectrometry Analysis ◽  
Confocal Laser ◽  
Steric Interaction ◽  
Content Type

ABSTRACT Pseudomonas aeruginosa plays a major role in many chronic infections. Its ability to readily form biofilms contributes to its success as an opportunistic pathogen and its resistance/tolerance to antimicrobial/antibiotic therapy. A low-molecular-weight alginate oligomer (OligoG CF-5/20) derived from marine algae has previously been shown to impair motility in P. aeruginosa biofilms and disrupt pseudomonal biofilm assembly. As these bacterial phenotypes are regulated by quorum sensing (QS), we hypothesized that OligoG CF-5/20 may induce alterations in QS signaling in P. aeruginosa . QS regulation was studied by using Chromobacterium violaceum CV026 biosensor assays that showed a significant reduction in acyl homoserine lactone (AHL) production following OligoG CF-5/20 treatment (≥2%; P < 0.05). This effect was confirmed by liquid chromatography-mass spectrometry analysis of C 4 -AHL and 3-oxo-C 12 -AHL production (≥2%; P < 0.05). Moreover, quantitative PCR showed that reduced expression of both the las and rhl systems was induced following 24 h of treatment with OligoG CF-5/20 (≥0.2%; P < 0.05). Circular dichroism spectroscopy indicated that these alterations were not due to steric interaction between the AHL and OligoG CF-5/20. Confocal laser scanning microscopy (CLSM) and COMSTAT image analysis demonstrated that OligoG CF-5/20-treated biofilms had a dose-dependent decrease in biomass that was associated with inhibition of extracellular DNA synthesis (≥0.5%; P < 0.05). These changes correlated with alterations in the extracellular production of the pseudomonal virulence factors pyocyanin, rhamnolipids, elastase, and total protease ( P < 0.05). The ability of OligoG CF-5/20 to modify QS signaling in P. aeruginosa PAO1 may influence critical downstream functions such as virulence factor production and biofilm formation.

scholarly journals Bacterial Swarming Reduces Proteus mirabilis and Vibrio parahaemolyticus Cell Stiffness and Increases β-Lactam Susceptibility

mBio ◽  
2019 ◽  
Vol 10 (5) ◽  
Author(s):  
George K. Auer ◽  
Piercen M. Oliver ◽  
Manohary Rajendram ◽  
Ti-Yu Lin ◽  
Qing Yao ◽  
...  
Keyword(s):  
Cell Wall ◽  
Osmotic Pressure ◽  
Proteus Mirabilis ◽  
Vibrio Parahaemolyticus ◽  
Cell Stiffness ◽  
Bending Rigidity ◽  
Vegetative Cells ◽  
Swarmer Cell ◽  
Content Type ◽  
Peptidoglycan Layer

ABSTRACT Swarmer cells of the Gram-negative uropathogenic bacteria Proteus mirabilis and Vibrio parahaemolyticus become long (>10 to 100 μm) and multinucleate during their growth and motility on polymer surfaces. We demonstrated that the increasing cell length is accompanied by a large increase in flexibility. Using a microfluidic assay to measure single-cell mechanics, we identified large differences in the swarmer cell stiffness (bending rigidity) of P. mirabilis (5.5 × 10−22 N m2) and V. parahaemolyticus (1.0 × 10−22 N m2) compared to vegetative cells (1.4 × 10−20 N m2 and 2.2 × 10−22 N m2, respectively). The reduction in bending rigidity (∼2-fold to ∼26-fold) was accompanied by a decrease in the average polysaccharide strand length of the peptidoglycan layer of the cell wall from 28 to 30 disaccharides to 19 to 22 disaccharides. Atomic force microscopy revealed a reduction in P. mirabilis peptidoglycan thickness from 1.5 nm (vegetative cells) to 1.0 nm (swarmer cells), and electron cryotomography indicated changes in swarmer cell wall morphology. P. mirabilis and V. parahaemolyticus swarmer cells became increasingly sensitive to osmotic pressure and susceptible to cell wall-modifying antibiotics (compared to vegetative cells)—they were ∼30% more likely to die after 3 h of treatment with MICs of the β-lactams cephalexin and penicillin G. The adaptive cost of “swarming” was offset by the increase in cell susceptibility to physical and chemical changes in their environment, thereby suggesting the development of new chemotherapies for bacteria that leverage swarming for the colonization of hosts and for survival. IMPORTANCE Proteus mirabilis and Vibrio parahaemolyticus are bacteria that infect humans. To adapt to environmental changes, these bacteria alter their cell morphology and move collectively to access new sources of nutrients in a process referred to as “swarming.” We found that changes in the composition and thickness of the peptidoglycan layer of the cell wall make swarmer cells of P. mirabilis and V. parahaemolyticus more flexible (i.e., reduce cell stiffness) and that they become more sensitive to osmotic pressure and cell wall-targeting antibiotics (e.g., β-lactams). These results highlight the importance of assessing the extracellular environment in determining antibiotic doses and the use of β-lactam antibiotics for treating infections caused by swarmer cells of P. mirabilis and V. parahaemolyticus.

scholarly journals Bacteriophage φEf11 ORF28 Endolysin, a Multifunctional Lytic Enzyme with Properties Distinct from All Other IdentifiedEnterococcus faecalisPhage Endolysins

2019 ◽  
Vol 85 (13) ◽  
Author(s):  
Hongming Zhang ◽  
Bettina A. Buttaro ◽  
Derrick E. Fouts ◽  
Salar Sanjari ◽  
Bradley S. Evans ◽  
...  
Keyword(s):  
Cell Wall ◽  
Enterococcus Faecalis ◽  
Amino Acid Identity ◽  
Lytic Infection ◽  
Mass Spectrometry Analysis ◽  
Lytic Enzyme ◽  
Lytic Enzymes ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Wide Range

ABSTRACTϕEf11 is a temperateSiphoviridaebacteriophage that infects strains ofEnterococcus faecalis. The ϕEf11 genome, encompassing 65 open reading frames (ORFs), is contained within 42,822 bp of DNA. Within this genome, a module of six lysis-related genes was identified. Based upon sequence homology, one of these six genes, ORF28, was predicted to code for anN-acetylmuramoyl-l-alanine amidase endolysin of 46.133 kDa, composed of 421 amino acids. The PCR-amplified ORF28 was cloned and expressed, and the resulting gene product was affinity purified to homogeneity. The purified protein was obtained from a fusion protein that exhibited a molecular mass of 72.5 kDa, consistent with a 46.1-kDa protein combined with a fused 26.5-kDa glutathioneS-transferase tag. It produced rapid, profound lysis inE. faecalispopulations and was active against 73 of 103 (71%)E. faecalisstrains tested. In addition, it caused substantial destruction ofE. faecalisbiofilms. The lysin was quite stable, retaining its activity for three years in refrigerated storage, was stable over a wide range of pHs, and was unaffected by the presence of a reducing agent; however, it was inhibited by increasing concentrations of Ca2+. Liquid chromatography-mass spectrometry analysis ofE. faecaliscell wall digestion products produced by the ORF28 endolysin indicated that the lysin acted as anN-acetylmuramidase, an endo-β-N-acetylglucosaminidase, and an endopeptidase, rather than anN-acetylmuramoyl-l-alanine amidase. The ϕEf11 ORF28 lysin shared 10% to 37% amino acid identity with the lytic enzymes of all other characterizedE. faecalisbacteriophages.IMPORTANCEThe emergence of multidrug-resistant pathogenic microorganisms has brought increasing attention to the urgent need for the development of alternative antimicrobial strategies. One such alternative to conventional antibiotics employs lytic enzymes (endolysins) that are produced by bacteriophages in the course of lytic infection. During lytic infection by a bacteriophage, these enzymes hydrolyze the cell wall peptidoglycan, resulting in the lysis of the host cell. However, external endolysin application can result in lysis from without. In this study, we have cloned, expressed, purified, and characterized an endolysin produced by a bacteriophage infecting strains ofEnterococcus faecalis. The lysin is broadly active against most of the testedE. faecalisstrains and exhibits multifunctional enzymatic specificities that differ from all other characterized endolysins produced byE. faecalisbacteriophages.

scholarly journals Role of the msaABCR Operon in Cell Wall Biosynthesis, Autolysis, Integrity, and Antibiotic Resistance in Staphylococcus aureus

2019 ◽  
Vol 63 (10) ◽  
Author(s):  
Bibek G C ◽  
Gyan S. Sahukhal ◽  
Mohamed O. Elasri
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Antibiotic Resistance ◽  
Teichoic Acid ◽  
Cross Linking ◽  
Wall Defect ◽  
Content Type ◽  
Mrsa Strains ◽  
Mutant Cells

ABSTRACT Staphylococcus aureus is an important human pathogen in both community and health care settings. One of the challenges with S. aureus as a pathogen is its acquisition of antibiotic resistance. Previously, we showed that deletion of the msaABCR operon reduces cell wall thickness, resulting in decreased resistance to vancomycin in vancomycin-intermediate S. aureus (VISA). In this study, we investigated the nature of the cell wall defect in the msaABCR operon mutant in the Mu50 (VISA) and USA300 LAC methicillin-resistant Staphylococcus aureus (MRSA) strains. Results showed that msaABCR mutant cells had decreased cross-linking in both strains. This defect is typically due to increased murein hydrolase activity and/or nonspecific processing of murein hydrolases mediated by increased protease activity in mutant cells. The defect was enhanced by a decrease in teichoic acid content in the msaABCR mutant. Therefore, we propose that deletion of the msaABCR operon results in decreased peptidoglycan cross-linking, leading to increased susceptibility toward cell wall-targeting antibiotics, such as β-lactams and vancomycin. Moreover, we also observed significantly downregulated transcription of early cell wall-synthesizing genes, supporting the finding that msaABCR mutant cells have decreased peptidoglycan synthesis. More specifically, the msaABCR mutant in the USA300 LAC strain (MRSA) showed significantly reduced expression of the murA gene, whereas the msaABCR mutant in the Mu50 strain (VISA) showed significantly reduced expression of glmU, murA, and murD. Thus, we conclude that the msaABCR operon controls the balance between cell wall synthesis and cell wall hydrolysis, which is required for maintaining a robust cell wall and acquiring resistance to cell wall-targeting antibiotics, such as vancomycin and the β-lactams.

scholarly journals Evidence that Biosynthesis of the Second and Third Sugars of the Archaellin Tetrasaccharide in the Archaeon Methanococcus maripaludis Occurs by the Same Pathway Used by Pseudomonas aeruginosa To Make a Di-N-Acetylated Sugar

2015 ◽  
Vol 197 (9) ◽  
pp. 1668-1680 ◽  
Author(s):  
Sarah Siu ◽  
Anna Robotham ◽  
Susan M. Logan ◽  
John F. Kelly ◽  
Kaoru Uchida ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Mass Spectroscopy ◽  
Specific Antigen ◽  
Mass Spectrometry Analysis ◽  
Methanococcus Maripaludis ◽  
Western Blots ◽  
Bacterial Gene ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Type Iv

ABSTRACTMethanococcus maripaludishas two surface appendages, archaella and type IV pili, which are composed of glycoprotein subunits. Archaellins are modified with an N-linked tetrasaccharide with the structure Sug-1,4-β-ManNAc3NAmA6Thr-1,4-β-GlcNAc3NAcA-1,3-β-GalNAc, where Sug is (5S)-2-acetamido-2,4-dideoxy-5-O-methyl-α-l-erythro-hexos-5-ulo-1,5-pyranose. The pilin glycan has an additional hexose attached to GalNAc. In this study, genes located in two adjacent, divergently transcribed operons (mmp0350-mmp0354andmmp0359-mmp0355) were targeted for study based on annotations suggesting their involvement in biosynthesis of N-glycan sugars. Mutants carrying deletions inmmp0350,mmp0351,mmp0352, ormmp0353were nonarchaellated and synthesized archaellins modified with a 1-sugar glycan, as estimated from Western blots. Mass spectroscopy analysis of pili purified from the Δmmp0352strain confirmed a glycan with only GalNAc, suggestingmmp0350tommp0353were all involved in biosynthesis of the second sugar (GlcNAc3NAcA). The Δmmp0357mutant was archaellated and had archaellins with a 2-sugar glycan, as confirmed by mass spectroscopy of purified archaella, indicating a role for MMP0357 in biosynthesis of the third sugar (ManNAc3NAmA6Thr).M. maripaludismmp0350,mmp0351,mmp0352,mmp0353, andmmp0357are proposed to be functionally equivalent toPseudomonas aeruginosawbpABEDI, involved in converting UDP-N-acetylglucosamine to UDP-2,3-diacetamido-2,3-dideoxy-d-mannuronic acid, an O5-specific antigen sugar. Cross-domain complementation of the final step of theP. aeruginosapathway withmmp0357supports this hypothesis.IMPORTANCEThis work identifies a series of genes in adjacent operons that are shown to encode the enzymes that complete the entire pathway for generation of the second and third sugars of the N-linked tetrasaccharide that modifies archaellins ofMethanococcus maripaludis. This posttranslational modification of archaellins is important, as it is necessary for archaellum assembly. Pilins are modified with a different N-glycan consisting of the archaellin tetrasaccharide but with an additional hexose attached to the linking sugar. Mass spectrometry analysis of the pili of one mutant strain provided insight into how this different glycan might ultimately be assembled. This study includes a rare example of an archaeal gene functionally replacing a bacterial gene in a complex sugar biosynthesis pathway.

scholarly journals High-Throughput Approaches for the Identification of Pseudomonas aeruginosa Antivirulents

mBio ◽  
2021 ◽  
Vol 12 (3) ◽  
Author(s):  
Donghoon Kang ◽  
Liyang Zhang ◽  
Natalia V. Kirienko
Keyword(s):  
Pseudomonas Aeruginosa ◽  
High Throughput ◽  
Bacterial Virulence ◽  
Special Focus ◽  
Content Type ◽  
Opportunistic Human Pathogen ◽  
New Treatments ◽  
High Throughput Screens ◽  
Synthetic Small Molecule ◽  
New Strategies

ABSTRACT Antimicrobial resistance is a serious medical threat, particularly given the decreasing rate of discovery of new treatments. Although attempts to find new treatments continue, it has become clear that merely discovering new antimicrobials, even if they are new classes, will be insufficient. It is essential that new strategies be aggressively pursued. Toward that end, the search for treatments that can mitigate bacterial virulence and tilt the balance of host-pathogen interactions in favor of the host has become increasingly popular. In this review, we will discuss recent progress in this field, with a special focus on synthetic small molecule antivirulents that have been identified from high-throughput screens and on treatments that are effective against the opportunistic human pathogen Pseudomonas aeruginosa.

scholarly journals The C Terminus of Substrates Is Critical but Not Sufficient for Their Degradation by the Pseudomonas aeruginosa CtpA Protease

2020 ◽  
Vol 202 (16) ◽  
Author(s):  
Sammi Chung ◽  
Andrew J. Darwin
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Cell Wall ◽  
Outer Membrane ◽  
Content Type ◽  
C Terminus ◽  
Outer Membrane Lipoprotein ◽  
Membrane Lipoprotein ◽  
Carboxyl Terminal

ABSTRACT Bacterial carboxyl-terminal processing proteases (CTPs) are widely conserved and have been linked to important processes, including signal transduction, cell wall metabolism, and virulence. However, the features that target proteins for CTP-dependent cleavage are unclear. Studies of the Escherichia coli CTP Prc suggested that it cleaves proteins with nonpolar and/or structurally unconstrained C termini, but it is not clear if this applies broadly. Pseudomonas aeruginosa has a divergent CTP, CtpA, which is required for virulence. CtpA works in complex with the outer membrane lipoprotein LbcA to degrade cell wall hydrolases. In this study, we investigated if the C termini of two nonhomologous CtpA substrates are important for their degradation. We determined that these substrates have extended C termini compared to those of their closest E. coli homologs. Removing 7 amino acids from these extensions was sufficient to reduce their degradation by CtpA both in vivo and in vitro. Degradation of one truncated substrate was restored by adding the C terminus from the other but not by adding an unrelated sequence. However, modification of the C termini of nonsubstrates, by adding the C-terminal amino acids from a substrate, did not cause their degradation by CtpA. Therefore, the C termini of CtpA substrates are required but not sufficient for their efficient degradation. Although C-terminal truncated substrates were protected from degradation, they still associated with the LbcA-CtpA complex in vivo. Therefore, degradation of a protein by CtpA requires a C terminus-independent interaction with the LbcA-CtpA complex, followed by C terminus-dependent degradation, perhaps because CtpA normally initiates cleavage at a C-terminal site. IMPORTANCE Carboxyl-terminal processing proteases (CTPs) are found in all three domains of life, but exactly how they work is poorly understood, including how they recognize substrates. Bacterial CTPs have been associated with virulence, including CtpA of Pseudomonas aeruginosa, which works in complex with the outer membrane lipoprotein LbcA to degrade potentially dangerous peptidoglycan hydrolases. We report an important advance by revealing that efficient degradation by CtpA requires at least two separable phenomena and that one of them depends on information encoded in the substrate C terminus. A C terminus-independent association with the LbcA-CtpA complex is followed by C terminus-dependent cleavage by CtpA. Increased understanding of how CTPs target proteins is significant, due to their links to virulence, peptidoglycan remodeling, and other important processes.

scholarly journals A Genome-WideHelicobacter pyloriMorphology Screen Uncovers a Membrane-Spanning Helical Cell Shape Complex

2019 ◽  
Vol 201 (14) ◽  
Author(s):  
Desirée C. Yang ◽  
Kris M. Blair ◽  
Jennifer A. Taylor ◽  
Timothy W. Petersen ◽  
Tate Sessler ◽  
...  
Keyword(s):  
Cell Wall ◽  
Helicobacter Pylori ◽  
Light Scattering ◽  
Protein Interactions ◽  
Cell Morphology ◽  
Cell Shape ◽  
Cross Linking ◽  
Protein Protein Interactions ◽  
Content Type ◽  
H Pylori

ABSTRACTEvident in its name, the gastric pathogenHelicobacter pylorihas a helical cell morphology which facilitates efficient colonization of the human stomach. An improved light-focusing strategy allowed us to robustly distinguish even subtle perturbations ofH. pyloricell morphology by deviations in light-scattering properties measured by flow cytometry. Profiling of an arrayed genome-wide deletion library identified 28 genes that influence different aspects of cell shape, including properties of the helix, cell length or width, cell filament formation, cell shape heterogeneity, and cell branching. Included in this mutant collection were two that failed to form any helical cells, a soluble lytic transglycosylase and a previously uncharacterized putative multipass inner membrane protein HPG27_0728, renamed Csd7. A combination of cell fractionation, mutational, and immunoprecipitation experiments show that Csd7 and Csd2 collaborate to stabilize the Csd1 peptidoglycan (PG) endopeptidase. Thus, bothcsd2andcsd7mutants show the same enhancement of PG tetra-pentapeptide cross-linking ascsd1mutants. Csd7 also links Csd1 with the bactofilin CcmA via protein-protein interactions. Although Csd1 is stable inccmAmutants, these mutants show altered PG tetra-pentapeptide cross-linking, suggesting that Csd7 may directly or indirectly activate as well as stabilize Csd1. These data begin to illuminate a highly orchestrated program to regulate PG modifications that promote helical shape, which includes nine nonessential nonredundant genes required for helical shape and 26 additional genes that further modifyH. pylori’s cell morphology.IMPORTANCEThe stomach ulcer and cancer-causing pathogenHelicobacter pylorihas a helical cell shape which facilitates stomach infection. Using light scattering to measure perturbations of cell morphology, we identified 28 genes that influence different aspects of cell shape. A mutant in a previously uncharacterized protein renamed Csd7 failed to form any helical cells. Biochemical analyses showed that Csd7 collaborates with other proteins to stabilize the cell wall-degrading enzyme Csd1. Csd7 also links Csd1 with a putative filament-forming protein via protein-protein interactions. These data suggest that helical cell shape arises from a highly orchestrated program to regulate cell wall modifications. Targeting of this helical cell shape-promoting program could offer new ways to block infectivity of this important human pathogen.

scholarly journals Ethanol Stimulates Trehalose Production through a SpoT-DksA-AlgU-Dependent Pathway inPseudomonas aeruginosa

2019 ◽  
Vol 201 (12) ◽  
Author(s):  
Colleen E. Harty ◽  
Dorival Martins ◽  
Georgia Doing ◽  
Dallas L. Mould ◽  
Michelle E. Clay ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Cell Density ◽  
Sigma Factor ◽  
Strong Support ◽  
Transcriptional Response ◽  
Exponential Phase ◽  
Fermentation Products ◽  
Content Type ◽  
In Cells

ABSTRACTPseudomonas aeruginosafrequently resides among ethanol-producing microbes, making its response to the microbially produced concentrations of ethanol relevant to understanding its biology. Our transcriptome analysis found that genes involved in trehalose metabolism were induced by low concentrations of ethanol, and biochemical assays showed that levels of intracellular trehalose increased significantly upon growth with ethanol. The increase in trehalose was dependent on the TreYZ pathway but not other trehalose-metabolic enzymes (TreS or TreA). The sigma factor AlgU (AlgT), a homolog of RpoE in other species, was required for increased expression of thetreZgene and trehalose levels, but induction was not controlled by the well-characterized proteolysis of its anti-sigma factor, MucA. Growth with ethanol led to increased SpoT-dependent (p)ppGpp accumulation, which stimulates AlgU-dependent transcription oftreZand other AlgU-regulated genes through DksA, a (p)ppGpp and RNA polymerase binding protein. Ethanol stimulation of trehalose also required acylhomoserine lactone (AHL)-mediated quorum sensing (QS), as induction was not observed in a ΔlasRΔrhlRstrain. A network analysis using a model, eADAGE, built from publicly availableP. aeruginosatranscriptome data sets (J. Tan, G. Doing, K. A. Lewis, C. E. Price, et al., Cell Syst 5:63–71, 2017, https://doi.org/10.1016/j.cels.2017.06.003) provided strong support for our model in whichtreZand coregulated genes are controlled by both AlgU- and AHL-mediated QS. Consistent with (p)ppGpp- and AHL-mediated quorum-sensing regulation, ethanol, even when added at the time of culture inoculation, stimulatedtreZtranscript levels and trehalose production in cells from post-exponential-phase cultures but not in cells from exponential-phase cultures. These data highlight the integration of growth and cell density cues in theP. aeruginosatranscriptional response to ethanol.IMPORTANCEPseudomonas aeruginosais often found with bacteria and fungi that produce fermentation products, including ethanol. At concentrations similar to those produced by environmental microbes, we found that ethanol stimulated expression of trehalose-biosynthetic genes and cellular levels of trehalose, a disaccharide that protects against environmental stresses. The induction of trehalose by ethanol required the alternative sigma factor AlgU through DksA- and SpoT-dependent (p)ppGpp. Trehalose accumulation also required AHL quorum sensing and occurred only in post-exponential-phase cultures. This work highlights how cells integrate cell density and growth cues in their responses to products made by other microbes and reveals a new role for (p)ppGpp in the regulation of AlgU activity.

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