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Journal of Bacteriology Sep 2022The Aer2 chemoreceptor from Pseudomonas aeruginosa is an O sensor involved in stress responses, virulence, and tuning the behavior of the chemotaxis (Che) system. Aer2...
The Aer2 chemoreceptor from Pseudomonas aeruginosa is an O sensor involved in stress responses, virulence, and tuning the behavior of the chemotaxis (Che) system. Aer2 is the sole receptor of the Che2 system. It is soluble, but membrane associated, and forms complexes at the cell pole during stationary phase. The domain arrangement of Aer2 is unusual, with a PAS sensing domain sandwiched between five HAMP domains, followed by a C-terminal kinase-control output domain. The first three HAMP domains form a poly-HAMP chain N-terminal to the PAS sensing domain. HAMP domains are often located between signal input and output domains, where they transduce signals. Given that HAMP1 to 3 reside N-terminal to the input-output pathway, we undertook a systematic examination of their function in Aer2. We found that HAMP1 to 3 influence PAS signaling over a considerable distance, as the majority of HAMP1, 2 and 3 mutations, and deletions of helical phase stutters, led to nonresponsive signal-off or off-biased receptors. PAS signal-on lesions that mimic activated Aer2 also failed to override N-terminal HAMP signal-off replacements. This indicates that HAMP1 to 3 are critical coupling partners for PAS signaling and likely function as a cohesive unit and moveable scaffold to correctly orient and poise PAS dimers for O-mediated signaling in Aer2. HAMP1 additionally controlled the clustering and polar localization of Aer2 in P. aeruginosa. Localization was not driven by HAMP1 charge, and HAMP1 signal-off mutants still localized. Employing HAMP as a clustering and localization determinant, as well as a facilitator of PAS signaling, are newly recognized roles for HAMP domains. P. aeruginosa is an opportunistic pathogen that interprets environmental stimuli via 26 chemoreceptors that signal through 4 distinct chemosensory systems. The second chemosensory system, Che2, contains a receptor named Aer2 that senses O and mediates stress responses and virulence and tunes chemotactic behavior. Aer2 is membrane associated, but soluble, and has three N-terminal HAMP domains (HAMP1 to 3) that reside outside the signal input-output pathway of Aer2. In this study, we determined that HAMP1 to 3 facilitate O-dependent signaling from the PAS sensing domain and that HAMP1 controls the formation of Aer2-containing polar foci in P. aeruginosa. Both of these are newly recognized roles for HAMP domains that may be applicable to other non-signal-transducing HAMP domains and poly-HAMP chains.
Topics: Carrier Proteins; Chemotaxis; Pseudomonas aeruginosa; Signal Transduction
PubMed: 35916529
DOI: 10.1128/jb.00225-22 -
Journal of Bacteriology Sep 2019frequently encounters microbes that produce ethanol. Low concentrations of ethanol reduced swim zone area by up to 45% in soft agar. The reduction of swimming by...
frequently encounters microbes that produce ethanol. Low concentrations of ethanol reduced swim zone area by up to 45% in soft agar. The reduction of swimming by ethanol required the flagellar motor proteins MotAB and two PilZ domain proteins (FlgZ and PilZ). PilY1 and the type 4 pilus alignment complex (comprising PilMNOP) were previously implicated in MotAB regulation in surface-associated cells and were required for ethanol-dependent motility repression. As FlgZ requires the second messenger bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) to represses motility, we screened mutants lacking genes involved in c-di-GMP metabolism and found that mutants lacking diguanylate cyclases SadC and GcbA were less responsive to ethanol. The double mutant was resistant to its effects. As published previously, ethanol also represses swarming motility, and the same genes required for ethanol effects on swimming motility were required for its regulation of swarming. Microscopic analysis of single cells in soft agar revealed that ethanol effects on swim zone area correlated with ethanol effects on the portion of cells that paused or stopped during the time interval analyzed. Ethanol increased c-di-GMP in planktonic wild-type cells but not in Δ or Δ Δ mutants, suggesting c-di-GMP plays a role in the response to ethanol in planktonic cells. We propose that ethanol produced by other microbes induces a regulated decrease in motility, thereby promoting colocalization with ethanol-producing microbes. Furthermore, some of the same factors involved in the response to surface contact are involved in the response to ethanol. Ethanol is an important biologically active molecule produced by many bacteria and fungi. It has also been identified as a potential marker for disease state in cystic fibrosis. In line with previous data showing that ethanol promotes biofilm formation by , here we report that ethanol reduces swimming motility using some of the same proteins involved in surface sensing. We propose that these data may provide insight into how microbes, via their metabolic byproducts, can influence colocalization in the context of infection and in other polymicrobial settings.
Topics: Bacterial Proteins; Ethanol; Flagella; Gene Expression Regulation, Bacterial; Movement; Pseudomonas aeruginosa
PubMed: 31109994
DOI: 10.1128/JB.00285-19 -
Applied and Environmental Microbiology Dec 2019Bacteria using toxic chemicals, such as detergents, as growth substrates face the challenge of exposing themselves to cell-damaging effects that require protection...
Bacteria using toxic chemicals, such as detergents, as growth substrates face the challenge of exposing themselves to cell-damaging effects that require protection mechanisms, which demand energy delivered from catabolism of the toxic compound. Thus, adaptations are necessary for ensuring the rapid onset of substrate degradation and the integrity of the cells. strain PAO1 can use the toxic detergent sodium dodecyl sulfate (SDS) as a growth substrate and employs, among others, cell aggregation as a protection mechanism. The degradation itself is also a protection mechanism and has to be rapidly induced upon contact to SDS. In this study, gene regulation of the enzymes initiating SDS degradation in strain PAO1 was studied. The gene and an atypical DNA-binding site of the LysR-type regulator SdsB1 were identified and shown to activate expression of the alkylsulfatase SdsA1 initiating SDS degradation. Further degradation of the resulting 1-dodecanol is catalyzed by enzymes encoded by , which were shown to form an operon. Expression of this operon is regulated by the TetR-type repressor LaoR. Studies with purified LaoR identified its DNA-binding site and 1-dodecanoyl coenzyme A as the ligand causing detachment of LaoR from the DNA. Transcriptional studies revealed that the sulfate ester detergent sodium lauryl ether sulfate (SLES) induced expression of and the operon. Growth experiments revealed an essential involvement of the alkylsulfatase SdsA1 for SLES degradation. This study revealed that the genes for the enzymes initiating the degradation of toxic sulfate-ester detergents are induced stepwise by a positive and a negative regulator in strain PAO1. Bacterial degradation of toxic compounds is important not only for bioremediation but also for the colonization of hostile anthropogenic environments in which biocides are being used. This study with expands our knowledge of gene regulation of the enzymes initiating degradation of sulfate ester detergents, which occurs in many hygiene and household products and, consequently, also in wastewater. As an opportunistic pathogen, causes severe hygienic problems because of its pronounced biocide resistance and its metabolic versatility, often combined with its pronounced biofilm formation. Knowledge about the regulation of detergent degradation, especially regarding the ligands of DNA-binding regulators, may lead to the rational development of specific inhibitors for restricting growth and biofilm formation of in hygienic settings. In addition, it may also contribute to optimizing bioremediation strategies not only for detergents but also for alkanes, which when degraded merge with sulfate ester degradation at the level of long-chain alcohols.
Topics: Esters; Gene Expression Regulation, Bacterial; Pseudomonas aeruginosa; Sodium Dodecyl Sulfate; Sulfatases; Sulfates
PubMed: 31540990
DOI: 10.1128/AEM.01352-19 -
Journal of Microbiology and... Jul 2015The emergence of carbapenem resistance among Pseudomonas aeruginosa is an increasing problem in many parts of the world. In particular, metallo-β-lactamases (MBLs) and...
The emergence of carbapenem resistance among Pseudomonas aeruginosa is an increasing problem in many parts of the world. In particular, metallo-β-lactamases (MBLs) and AmpC β- lactamases are responsible for high-level resistance to carbapenem and cephalosporin. We studied the diversity and frequency of β-lactamases and characterized chromosomal AmpC β- lactamase from carbapenem-resistant P. aeruginosa isolates. Sixty-one carbapenem-resistant P. aeruginosa isolates were collected from patients in a tertiary hospital in Daejeon, Korea, from January 2011 to June 2014. Minimum inhibitory concentrations (MICs) of four antimicrobial agents were determined using the agar-dilution method. Polymerase chain reaction and sequencing were used to identify the various β-lactamase genes, class 1 integrons, and chromosomally encoded and plasmid-mediated ampC genes. In addition, the epidemiological relationship was investigated by multilocus sequence typing. Among 61 carbapenem-resistant P. aeruginosa isolates, 25 isolates (41.0%) were MBL producers. Additionally, 30 isolates producing PDC (Pseudomonas-derived cephalosporinase)-2 were highly resistant to ceftazidime (MIC50 = 256 μg/ml) and cefepime (MIC50 = 256 μg/ml). Of all the PDC variants, 25 isolates harboring MBL genes showed high levels of cephalosporin and carbapenem resistance, whereas 36 isolates that did not harbor MBL genes revealed relatively low-level resistance (ceftazidime, p < 0.001; cefepime, p < 0.001; imipenem, p = 0.003; meropenem, p < 0.001). The coexistence of MBLs and AmpC β-lactamases suggests that these may be important contributing factors for cephalosporin and carbapenem resistance. Therefore, efficient detection and intervention to control drug resistance are necessary to prevent the emergence of P. aeruginosa possessing this combination of β-lactamases.
Topics: Anti-Bacterial Agents; Bacterial Proteins; Carbapenems; Cephalosporinase; Genetic Variation; Genotype; Humans; Microbial Sensitivity Tests; Multilocus Sequence Typing; Polymerase Chain Reaction; Pseudomonas Infections; Pseudomonas aeruginosa; Republic of Korea; Sequence Analysis, DNA; Tertiary Care Centers; beta-Lactam Resistance; beta-Lactamases
PubMed: 25907063
DOI: 10.4014/jmb.1503.03065 -
MBio Sep 2015Pulmonary infections caused by Pseudomonas aeruginosa are a recalcitrant problem in cystic fibrosis (CF) patients. While the clinical implications and long-term...
UNLABELLED
Pulmonary infections caused by Pseudomonas aeruginosa are a recalcitrant problem in cystic fibrosis (CF) patients. While the clinical implications and long-term evolutionary patterns of these infections are well studied, we know little about the short-term population dynamics that enable this pathogen to persist despite aggressive antimicrobial therapy. Here, we describe a short-term population genomic analysis of 233 P. aeruginosa isolates collected from 12 sputum specimens obtained over a 1-year period from a single patient. Whole-genome sequencing and antimicrobial susceptibility profiling identified the expansion of two clonal lineages. The first lineage originated from the coalescence of the entire sample less than 3 years before the end of the study and gave rise to a high-diversity ancestral population. The second expansion occurred 2 years later and gave rise to a derived population with a strong signal of positive selection. These events show characteristics consistent with recurrent selective sweeps. While we cannot identify the specific mutations responsible for the origins of the clonal lineages, we find that the majority of mutations occur in loci previously associated with virulence and resistance. Additionally, approximately one-third of all mutations occur in loci that are mutated multiple times, highlighting the importance of parallel pathoadaptation. One such locus is the gene encoding penicillin-binding protein 3, which received three independent mutations. Our functional analysis of these alleles shows that they provide differential fitness benefits dependent on the antibiotic under selection. These data reveal that bacterial populations can undergo extensive and dramatic changes that are not revealed by lower-resolution analyses.
IMPORTANCE
Pseudomonas aeruginosa is a bacterial opportunistic pathogen responsible for significant morbidity and mortality in cystic fibrosis (CF) patients. Once it has colonized the lung in CF, it is highly resilient and rarely eradicated. This study presents a deep sampling examination of the fine-scale evolutionary dynamics of P. aeruginosa in the lungs of a chronically infected CF patient. We show that diversity of P. aeruginosa is driven by recurrent clonal emergence and expansion within this patient and identify potential adaptive variants associated with these events. This high-resolution sequencing strategy thus reveals important intraspecies dynamics that explain a clinically important phenomenon not evident at a lower-resolution analysis of community structure.
Topics: Adaptation, Biological; Biota; Cluster Analysis; Cystic Fibrosis; DNA, Bacterial; Evolution, Molecular; Genetic Variation; Genetics, Population; Genome, Bacterial; Genotype; Humans; Lung; Pseudomonas Infections; Pseudomonas aeruginosa; Selection, Genetic; Sequence Analysis, DNA; Sequence Homology; Sputum
PubMed: 26330513
DOI: 10.1128/mBio.00981-15 -
Emerging Microbes & Infections Dec 2021is a clinically important pathogen implicated in many hospital-acquired infections. Its propensity to acquire broad-spectrum resistance has earned the organism its...
is a clinically important pathogen implicated in many hospital-acquired infections. Its propensity to acquire broad-spectrum resistance has earned the organism its status as a severe public health threat requiring urgent control measures. While whole-genome sequencing-based genomic surveillance provides a means to track antimicrobial resistance, its use in molecular epidemiological surveys of remains limited, especially in the Southeast Asian region. We sequenced the whole genomes of 222 carbapenem-non-susceptible (CNPA) isolates collected in 2006-2020 at the largest public acute care hospital in Singapore. Antimicrobial susceptibilities were determined using broth microdilution. Clonal relatedness, multi-locus sequence types, and antimicrobial resistance determinants (acquired and chromosomal) were determined. In this study, CNPA exhibited broad-spectrum resistance (87.8% multi-drug resistance), retaining susceptibility only to polymyxin B (95.0%) and amikacin (55.0%). Carbapenemases were detected in 51.4% of the isolates, where IMP and NDM metallo--lactamases were the most frequent. Carbapenem resistance was also likely associated with OprD alterations or efflux mechanisms (ArmZ/NalD mutations), which occurred in strains with or without carbapenemases. The population of CNPA in the hospital was diverse; the 222 isolates grouped into 68 sequence types (ST), which included various high-risk clones. We detected an emerging clone, the NDM-1-producing ST308, in addition to the global high-risk ST235 clone which was the predominant clone in our population. Our results thus provide a "snapshot" of the circulating lineages of CNPA locally and the prevailing genetic mechanisms contributing to carbapenem resistance. This database also serves as the baseline for future prospective surveillance studies.
Topics: Anti-Bacterial Agents; Bacterial Typing Techniques; Carbapenems; Drug Resistance, Bacterial; Genome, Bacterial; Genomics; Humans; Microbial Sensitivity Tests; Multilocus Sequence Typing; Prospective Studies; Pseudomonas aeruginosa; Singapore; Whole Genome Sequencing
PubMed: 34384341
DOI: 10.1080/22221751.2021.1968318 -
The American Naturalist Sep 2019Predicting the evolution of expanding populations is critical to controlling biological threats such as invasive species and cancer metastasis. Expansion is primarily...
Predicting the evolution of expanding populations is critical to controlling biological threats such as invasive species and cancer metastasis. Expansion is primarily driven by reproduction and dispersal, but nature abounds with examples of evolution where organisms pay a reproductive cost to disperse faster. When does selection favor this "survival of the fastest"? We searched for a simple rule, motivated by evolution experiments where swarming bacteria evolved into a hyperswarmer mutant that disperses ∼100% faster but pays a growth cost of ∼10% to make many copies of its flagellum. We analyzed a two-species model based on the Fisher equation to explain this observation: the population expansion rate () results from an interplay of growth () and dispersal () and is independent of the carrying capacity: . A mutant can take over the edge only if its expansion rate () exceeds the expansion rate of the established species (); this simple condition ( ) determines the maximum cost in slower growth that a faster mutant can pay and still be able to take over. Numerical simulations and time-course experiments where we tracked evolution by imaging bacteria suggest that our findings are general: less favorable conditions delay but do not entirely prevent the success of the fastest. Thus, the expansion rate defines a traveling wave fitness, which could be combined with trade-offs to predict evolution of expanding populations.
Topics: Biological Evolution; Green Fluorescent Proteins; Models, Theoretical; Mutation; Pseudomonas aeruginosa; Selection, Genetic
PubMed: 31553215
DOI: 10.1086/704594 -
Journal of Virology Aug 2015Antimicrobial resistance constitutes one of the major worldwide public health concerns. Bacteria are becoming resistant to the vast majority of antibiotics, and... (Review)
Review
Antimicrobial resistance constitutes one of the major worldwide public health concerns. Bacteria are becoming resistant to the vast majority of antibiotics, and nowadays, a common infection can be fatal. To address this situation, the use of phages for the treatment of bacterial infections has been extensively studied as an alternative therapeutic strategy. Since Pseudomonas aeruginosa is one of the most common causes of health care-associated infections, many studies have reported the in vitro and in vivo antibacterial efficacy of phage therapy against this bacterium. This review collects data of all the P. aeruginosa phages sequenced to date, providing a better understanding about their biodiversity. This review further addresses the in vitro and in vivo results obtained by using phages to treat or prevent P. aeruginosa infections as well as the major hurdles associated with this therapy.
Topics: Animals; Biological Therapy; Disease Models, Animal; Humans; Pseudomonas Infections; Pseudomonas Phages; Pseudomonas aeruginosa
PubMed: 25972556
DOI: 10.1128/JVI.00385-15 -
Proceedings of the National Academy of... Feb 2015The bacterium Pseudomonas aeruginosa is an opportunistic human pathogen that uses a quorum sensing signal cascade to activate expression of dozens of genes when...
The bacterium Pseudomonas aeruginosa is an opportunistic human pathogen that uses a quorum sensing signal cascade to activate expression of dozens of genes when sufficient population densities have been reached. Quorum sensing controls production of several key virulence factors, including secreted proteases such as elastase. Cooperating groups of bacteria growing on protein are susceptible to social cheating by quorum-sensing defective mutants. A possible way to restrict cheater emergence is by policing where cooperators produce costly goods to sanction or punish cheats. The P. aeruginosa LasR-LasI quorum sensing system controls genes including those encoding proteases and also those encoding a second quorum-sensing system, the RhlR-RhlI system, which controls numerous genes including those for cyanide production. By using RhlR quorum sensing mutants and cyanide synthesis mutants, we show that cyanide production is costly and cyanide-producing cooperators use cyanide to punish LasR-null social cheaters. Cooperators are less susceptible to cyanide than are LasR mutants. These experiments demonstrate policing in P. aeruginosa, provide a mechanistic understanding of policing, and show policing involves the cascade organization of the two quorum sensing systems in this bacterium.
Topics: Cyanides; Mutation; Pseudomonas aeruginosa; Quorum Sensing; Virulence
PubMed: 25646454
DOI: 10.1073/pnas.1500704112 -
Journal of Medical Microbiology Jan 2020Laboratory research with commonly involves the prototype strain PAO1. There is continued concern that PAO1 sublines maintained and propagated in the same laboratory or...
Laboratory research with commonly involves the prototype strain PAO1. There is continued concern that PAO1 sublines maintained and propagated in the same laboratory or different laboratories exhibit genetic and phenotypic variability that may affect the reproducibility and validity of research. Whole-genome sequencing and other research identified the locus as a mutational hotspot, but the explication of the diverse mutations present in the various sublines and consequences remained rather cursory. Here we present evidence that MexT sequence diversity is a predictor of PAO1 lineage integrity and define the protein's prototype sequence.
Topics: Bacterial Proteins; Genetic Variation; Mutation; Pseudomonas aeruginosa; Serial Passage; Whole Genome Sequencing
PubMed: 31859619
DOI: 10.1099/jmm.0.001128