PsrA controls the synthesis of the Pseudomonas aeruginosa quinolone signal via repression of the FadE homolog, PA0506

Pseudomonas aeruginosa is a ubiquitous, Gram-negative opportunistic pathogen that can cause disease in various sites within the human body. This bacterium is a major source of nosocomial infections that are often difficult to treat due to high intrinsic antibiotic resistance and coordinated virulence factor production. P. aeruginosa utilizes three cell-to-cell signal- ing systems to regulate numerous genes in response to cell density. One of these systems utilizes the small molecule 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas quinolone signal [PQS]) as a signal that acts as a co-inducer for the transcriptional regulator PqsR. Quinolone signaling is required for virulence in multiple infection models, and PQS is produced during human infections, making this system an attractive target for potential drug development. In this study we have examined the role of a TetR-type transcriptional regulator, PsrA, in the regulation of PQS production by P. aeruginosa. Previous studies showed that PsrA regu- lates genes of the fatty acid β-oxidation pathway, including PA0506, which encodes a FadE homolog. In this report, we show that deletion of psrA resulted in a large decrease in PQS production and that co-deletion of PA0506 allowed PQS production to be restored to a wild type level. We also found that PQS production could be restored to the psrA mutant by the addition of oleic or octanoic acid. Taken together, our data suggest that psrA positively affects PQS production by repressing the transcription of PA0506, which leads to a decrease in the conversion of acyl-CoA compounds to enoyl-CoA compounds, thereby allowing some octanoyl-CoA to escape the ß-oxidation pathway and serve as a PQS precursor

Acinetobacter baumannii Regulates Its Stress Responses via the BfmRS Two-Component Regulatory System

Acinetobacter baumannii is a common nosocomial pathogen that utilizes numerous mechanisms to aid its survival in both the environment and the host. Coordination of such mechanisms requires an intricate regulatory network. We report here that A. baumannii can directly regulate several stress-related pathways via the two-component regulatory system BfmRS. Similar to previous studies, results from transcriptomic analysis showed that mutation of the BfmR response regulator causes dysregulation of genes required for the oxidative stress response, the osmotic stress response, the misfolded protein/heat shock response, Csu pilus/fimbria production, and capsular polysaccharide biosynthesis. We also found that the BfmRS system is involved in controlling siderophore biosynthesis and transport, and type IV pili production. We provide evidence that BfmR binds to various stress-related promoter regions and show that BfmR alone can directly activate transcription of some stress-related genes. Additionally, we show that the BfmS sensor kinase acts as a BfmR phosphatase to negatively regulate BfmR activity. This work highlights the importance of the BfmRS system in promoting survival of A. baumannii. IMPORTANCE Acinetobacter baumannii is a nosocomial pathogen that has extremely high rates of multidrug resistance. This organism’s ability to endure stressful conditions is a key part of its ability to spread in the hospital environment and cause infections. Unlike other members of the gammaproteobacteria, A. baumannii does not encode a homolog of the RpoS sigma factor to coordinate its stress response. Here, we demonstrate that the BfmRS two-component system directly controls the expression of multiple stress resistance genes. Our findings suggest that BfmRS is central to a unique scheme of general stress response regulation by A. baumannii

The respective roles of BfmRS and PmrA in stress responses and antibiotic resistance in Acinetobacter baumannii

The Gram-negative bacterium Acinetobacter baumannii is considered one of the most serious nosocomial pathogens worldwide. Predominantly responsible for ventilator-associated pneumonia, this insidious pathogen typically infects critically ill individuals and patients in long-term care. A. baumannii harbors a myriad of resistance mechanisms to aid its survival in both the environment and the host. Remarkably, A. baumannii does not encode an RpoS sigma factor homolog that is used to control the general stress response by the majority of Gram-negative bacteria. Instead, A. baumannii relies on an intricate regulatory network involving numerous two-component regulatory systems to respond to stress. Two key players that contribute to A. baumannii’s “persist and resist� survival mechanisms are the BfmRS and PmrAB two-component regulatory systems. We have demonstrated that the response regulator BfmR can directly activate numerous stress-related pathways. We also provide evidence that the sensor kinase BfmS acts as a phosphatase to negatively regulate BfmR activity. Overall, we show that the BfmRS system harbors the characteristics of a master regulator by controlling the osmotic stress response, the oxidative stress response, the misfolded protein response, csu pili/fimbriae production, capsule polysaccharide biosynthesis, siderophore biosynthesis and transport, type IV pili production, and antibiotic resistance. Additionally, the PmrAB two-component system contributes to antibiotic resistance in A. baumannii. Clinically relevant mutations that arise in the pmrCAB operon often promote resistance to the last resort antibiotic colistin. We biochemically characterized the response regulator PmrA to identify its DNA-binding domain, in addition to the potential PmrA regulon. We also provide the first structural information for the PmrA N-terminal domain. Together, these data allowed us to analyze the structural and dynamic changes in two clinically relevant PmrA point mutants that alter the function of PmrA and promote colistin resistance. Understanding these regulatory mechanisms at a molecular level will allow us to explore novel ways to develop effective antimicrobial agents to combat this serious pathogen

Phonetic archaeology and 50 years of change to Australian English

The work reported here explores the issue of Australian English accent broadness, past and present, through a diachronic acoustic analysis of the vowel,drawing on archival data collected by Mitchell and Delbridge in the late 1950s and early 1960s and more recent data from the Australian Voices project. Data from 168 female speakers from the Mitchell and Delbridge survey and 70 female speakers from the Australian Voices project were examined. All were from Sydney's North and North West and represented the Government, Catholic and Independent school systems. A number of acoustic measurements were employed to identify variation and change associated with this vowel extracted from a single word in a sentence reading task. In particular, we were interested in the degree of onglide, a feature of that is pervasive in Australian English. We provide empirical evidence showing that the broadness continuum has contracted by demonstrating that variation in the degree of onglide for has changed in interesting ways for girls from three different school systems.26 page(s

Hiatus resolution and linking 'r' in Australian English

Hiatus occurs when the juxtaposition of syllables results in two separate vowels occurring alongside one another. Such vowel adjacency, both within words and across word boundaries, is phonologically undesirable in many languages but can be resolved using a range of strategies including consonant insertion. This paper examines linguistic and extralinguistic factors that best predict the likelihood of inserted linking 'r' across word boundaries in Australian English. Corpus data containing a set of 32 phrases produced in a sentence-reading task by 103 speakers were auditorily and acoustically analysed. Results reveal that linguistic variables of accentual context and local speaking rate take precedence over speaker-specific variables of age, gender and sociolect in the management of hiatus. We interpret this to be a reflection of the phonetic manifestation of boundary phenomena. The frequency of the phrase containing the linking 'r', the frequency of an individual's use of linking 'r', and the accentual status of the flanking vowels all affect the/ɺ/strength (determined by F3), suggesting that a hybrid approach is warranted in modelling liaison. Age effects are present for certain prosodic contexts indicating change in progress for Australian English.24 page(s

The respective roles of BfmRS and PmrA in stress responses and antibiotic resistance in Acinetobacter baumannii

The Gram-negative bacterium Acinetobacter baumannii is considered one of the most serious nosocomial pathogens worldwide. Predominantly responsible for ventilator-associated pneumonia, this insidious pathogen typically infects critically ill individuals and patients in long-term care. A. baumannii harbors a myriad of resistance mechanisms to aid its survival in both the environment and the host. Remarkably, A. baumannii does not encode an RpoS sigma factor homolog that is used to control the general stress response by the majority of Gram-negative bacteria. Instead, A. baumannii relies on an intricate regulatory network involving numerous two-component regulatory systems to respond to stress. Two key players that contribute to A. baumannii's "persist and resist" survival mechanisms are the BfmRS and PmrAB two-component regulatory systems. We have demonstrated that the response regulator BfmR can directly activate numerous stress-related pathways. We also provide evidence that the sensor kinase BfmS acts as a phosphatase to negatively regulate BfmR activity. Overall, we show that the BfmRS system harbors the characteristics of a master regulator by controlling the osmotic stress response, the oxidative stress response, the misfolded protein response, csu pili/fimbriae production, capsule polysaccharide biosynthesis, siderophore biosynthesis and transport, type IV pili production, and antibiotic resistance. Additionally, the PmrAB two-component system contributes to antibiotic resistance in A. baumannii. Clinically relevant mutations that arise in the pmrCAB operon often promote resistance to the last resort antibiotic colistin. We biochemically characterized the response regulator PmrA to identify its DNA-binding domain, in addition to the potential PmrA regulon. We also provide the first structural information for the PmrA N-terminal domain. Together, these data allowed us to analyze the structural and dynamic changes in two clinically relevant PmrA point mutants that alter the function of PmrA and promote colistin resistance. Understanding these regulatory mechanisms at a molecular level will allow us to explore novel ways to develop effective antimicrobial agents to combat this serious pathogen

PsrA controls the synthesis of the <i>Pseudomonas aeruginosa</i> quinolone signal via repression of the FadE homolog, PA0506

<div><p><i>Pseudomonas aeruginosa</i> is a ubiquitous, Gram-negative opportunistic pathogen that can cause disease in various sites within the human body. This bacterium is a major source of nosocomial infections that are often difficult to treat due to high intrinsic antibiotic resistance and coordinated virulence factor production. <i>P</i>. <i>aeruginosa</i> utilizes three cell-to-cell signaling systems to regulate numerous genes in response to cell density. One of these systems utilizes the small molecule 2-heptyl-3-hydroxy-4-quinolone (<i>Pseudomonas</i> quinolone signal [PQS]) as a signal that acts as a co-inducer for the transcriptional regulator PqsR. Quinolone signaling is required for virulence in multiple infection models, and PQS is produced during human infections, making this system an attractive target for potential drug development. In this study we have examined the role of a TetR-type transcriptional regulator, PsrA, in the regulation of PQS production by <i>P</i>. <i>aeruginosa</i>. Previous studies showed that PsrA regulates genes of the fatty acid β-oxidation pathway, including <i>PA0506</i>, which encodes a FadE homolog. In this report, we show that deletion of <i>psrA</i> resulted in a large decrease in PQS production and that co-deletion of <i>PA0506</i> allowed PQS production to be restored to a wild type level. We also found that PQS production could be restored to the <i>psrA</i> mutant by the addition of oleic or octanoic acid. Taken together, our data suggest that <i>psrA</i> positively affects PQS production by repressing the transcription of <i>PA0506</i>, which leads to a decrease in the conversion of acyl-CoA compounds to enoyl-CoA compounds, thereby allowing some octanoyl-CoA to escape the ß-oxidation pathway and serve as a PQS precursor.</p></div

PsrA positively controls <i>pqsA</i>.

<p>(A) qRT-PCR was performed on RNA from strains PAO1 and PGW-Δ<i>psrA</i> to analyze the relative expression of <i>lasR</i>, <i>pqsH</i>, <i>pqsR</i> and <i>pqsA</i>. Data are presented as average fold change ± SD in the <i>psrA</i> mutant compared to the wild type strain (set to a value of 1). The gene <i>clpX</i> was used as a reference gene to normalize expression and each experiment was completely repeated three times. (B and C) PQS production by strains PAO1 or PGW-Δ<i>psrA</i> expressing (B) PQS synthetic genes or (C) RpoS. Cultures were grown for 24 h in LB medium supplemented with 0.5% L-arabinose to induce genes. PQS was then extracted and quantified as described in Materials and Methods. Data are presented as the average ± SD of three independent experiments. (B) Plasmids contained by strains are: open bars, control vector; solid bars, <i>pqsABCD</i> expression vector; and hatched bars, <i>pqsH</i> expression vector. (C) Presence of the <i>rpoS</i> expression vector is indicated by a plus (+) symbol.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

Increased expression of <i>PA0506</i> alters PQS production.

<p>The indicated strains were grown for 24 h in LB medium harboring pHERD20T (control plasmid) or pGW-21 (<i>PA0506</i> expression plasmid) indicated by a minus or plus symbol, respectively. Cultures were supplemented with 0.5% L-arabinose to induce <i>PA0506</i> expression, and PQS was then extracted and quantified. Data are presented as the average ± SD of three independent experiments.</p