scholarly journals Catalase-Peroxidases of Legionella pneumophila: Cloning of the katA Gene and Studies of KatA Function

2000 ◽  
Vol 182 (23) ◽  
pp. 6679-6686 ◽  
Author(s):  
Purnima Bandyopadhyay ◽  
Howard M. Steinman
Keyword(s):  
Hydrogen Peroxide ◽  
Stationary Phase ◽  
Legionella Pneumophila ◽  
Redox Reactions ◽  
Causative Organism ◽  
Wild Type ◽  
Macrophage Cell ◽  
Peroxidatic Activity ◽  
Stationary Phase Survival

ABSTRACT Legionella pneumophila, the causative organism of Legionnaires' pneumonia, contains two enzymes with catalatic and peroxidatic activity, KatA and KatB. To address the issue of redundant, overlapping, or discrete in vivo functions of highly homologous catalase-peroxidases, the gene for katA was cloned and its function was studied in L. pneumophila andEscherichia coli and compared with prior studies ofkatB in this laboratory. katA is induced during exponential growth and is the predominant peroxidase in stationary phase. When katA is inactivated, L. pneumophilais more sensitive to exogenous hydrogen peroxide and less virulent in the THP-1 macrophage cell line, similar to katB. Catalatic-peroxidatic activity with different peroxidatic cosubstrates is comparable for KatA and KatB, but KatA is five times more active towards dianisidine. In contrast with these examples of redundant or overlapping function, stationary-phase survival is decreased by 100- to 10,000-fold when katA is inactivated, while no change from wild type is seen for the katB null. The principal clue for understanding this discrete in vivo function was the demonstration that KatA is periplasmic and KatB is cytosolic. This stationary-phase phenotype suggests that targets sensitive to hydrogen peroxide are present outside the cytosol in stationary phase or that the peroxidatic activity of KatA is critical for stationary-phase redox reactions in the periplasm, perhaps disulfide bond formation. Since starvation-induced stationary phase is a prerequisite to acquisition of virulence by L. pneumophila, further studies on the function and regulation of katA in stationary phase may give insights on the mechanisms of infectivity of this pathogen.

scholarly journals Effects of Combination of Different −10 Hexamers and Downstream Sequences on Stationary-Phase-Specific Sigma Factor ςS-Dependent Transcription in Pseudomonas putida

2000 ◽  
Vol 182 (23) ◽  
pp. 6707-6713 ◽  
Author(s):  
Eve-Ly Ojangu ◽  
Andres Tover ◽  
Riho Teras ◽  
Maia Kivisaar
Keyword(s):  
Stationary Phase ◽  
Pseudomonas Putida ◽  
Sigma Factor ◽  
Sigma Factors ◽  
Deficient Mutant ◽  
Wild Type ◽  
In Vivo Experiments ◽  
Silent Genes ◽  
Rna Polymerase Holoenzyme

ABSTRACT The main sigma factor activating gene expression, necessary in stationary phase and under stress conditions, is ςS. In contrast to other minor sigma factors, RNA polymerase holoenzyme containing ςS (EςS) recognizes a number of promoters which are also recognized by that containing ς70 (Eς70). We have previously shown that transposon Tn4652 can activate silent genes in starvingPseudomonas putida cells by creating fusion promoters during transposition. The sequence of the fusion promoters is similar to the ς70-specific promoter consensus. The −10 hexameric sequence and the sequence downstream from the −10 element differ among these promoters. We found that transcription from the fusion promoters is stationary phase specific. Based on in vivo experiments carried out with wild-type and rpoS-deficient mutant P. putida, the effect of ςS on transcription from the fusion promoters was established only in some of these promoters. The importance of the sequence of the −10 hexamer has been pointed out in several published papers, but there is no information about whether the sequences downstream from the −10 element can affect ςS-dependent transcription. Combination of the −10 hexameric sequences and downstream sequences of different fusion promoters revealed that ςS-specific transcription from these promoters is not determined by the −10 hexameric sequence only. The results obtained in this study indicate that the sequence of the −10 element influences ςS-specific transcription in concert with the sequence downstream from the −10 box.

scholarly journals Staphylococcus aureus Aconitase Inactivation Unexpectedly Inhibits Post-Exponential-Phase Growth and Enhances Stationary-Phase Survival

2002 ◽  
Vol 70 (11) ◽  
pp. 6373-6382 ◽  
Author(s):  
Greg A. Somerville ◽  
Michael S. Chaussee ◽  
Carrie I. Morgan ◽  
J. Ross Fitzgerald ◽  
David W. Dorward ◽  
...  
Keyword(s):  
Stationary Phase ◽  
Tca Cycle ◽  
Exponential Growth Phase ◽  
Wild Type ◽  
Phase Growth ◽  
Iron Sulfur Cluster ◽  
Iron Sulfur ◽  
The Tca Cycle ◽  
Stationary Phase Survival ◽  
Virulence Factor Production

ABSTRACT Staphylococcus aureus preferentially catabolizes glucose, generating pyruvate, which is subsequently oxidized to acetate under aerobic growth conditions. Catabolite repression of the tricarboxylic acid (TCA) cycle results in the accumulation of acetate. TCA cycle derepression coincides with exit from the exponential growth phase, the onset of acetate catabolism, and the maximal expression of secreted virulence factors. These data suggest that carbon and energy for post-exponential-phase growth and virulence factor production are derived from the catabolism of acetate mediated by the TCA cycle. To test this hypothesis, the aconitase gene was genetically inactivated in a human isolate of S. aureus, and the effects on physiology, morphology, virulence factor production, virulence for mice, and stationary-phase survival were examined. TCA cycle inactivation prevented the post-exponential growth phase catabolism of acetate, resulting in premature entry into the stationary phase. This phenotype was accompanied by a significant reduction in the production of several virulence factors and alteration in host-pathogen interaction. Unexpectedly, aconitase inactivation enhanced stationary-phase survival relative to the wild-type strain. Aconitase is an iron-sulfur cluster-containing enzyme that is highly susceptible to oxidative inactivation. We speculate that reversible loss of the iron-sulfur cluster in wild-type organisms is a survival strategy used to circumvent oxidative stress induced during host-pathogen interactions. Taken together, these data demonstrate the importance of the TCA cycle in the life cycle of this medically important pathogen.

scholarly journals Characterization of a Legionella pneumophila relA Insertion Mutant and Roles of RelA and RpoS in Virulence Gene Expression

2002 ◽  
Vol 184 (1) ◽  
pp. 67-75 ◽  
Author(s):  
Tal Zusman ◽  
Ohad Gal-Mor ◽  
Gil Segal
Keyword(s):  
Gene Expression ◽  
Stationary Phase ◽  
Gene Product ◽  
Legionella Pneumophila ◽  
Type Strain ◽  
Wild Type Strain ◽  
Insertion Mutant ◽  
Wild Type ◽  
Intracellular Growth ◽  
Virulence Gene Expression

ABSTRACT To investigate the involvement of RelA in the regulation of Legionella pneumophila virulence, a deletion substitution was constructed in the relA gene. The relA knockout resulted in an undetectable level of ppGpp in the cells during the stationary phase, but the original level was restored when the relA gene product was supplied on a plasmid. The effect of the relA mutation was examined with two systems that are known to be expressed during the stationary phase in L. pneumophila. Pigment production was found to be dependent on the relA gene product, and only one-half as much pigment was produced by the relA mutant as by the wild-type strain. Flagellum gene expression was also found to be dependent on the relA gene product, as determined with a flaA::lacZ fusion. However, the relA gene product was found to be dispensable for intracellular growth both in HL-60-derived human macrophages and in the protozoan host Acanthamoeba castellanii. To determine the involvement of the relA gene product in expression of L. pneumophila genes required for intracellular growth (icm/dot genes), nine icm::lacZ fusions were constructed, and expression of these fusions in the wild-type strain was compared with their expression in relA mutant strains. Expression of only one of the icm::lacZ fusions was moderately reduced in the relA mutant strain. Expression of the nine icm::lacZ fusions was also examined in a strain containing an insertion in the gene that codes for the stationary-phase sigma factor RpoS, and similar results were obtained. We concluded that RelA is dispensable for intracellular growth of L. pneumophila in the two hosts examined and that both RelA and RpoS play minor roles in L. pneumophila icm/dot gene expression.

scholarly journals The Legionella pneumophila rpoS Gene Is Required for Growth within Acanthamoeba castellanii

1999 ◽  
Vol 181 (16) ◽  
pp. 4879-4889 ◽  
Author(s):  
Laura M. Hales ◽  
Howard A. Shuman
Keyword(s):  
Stationary Phase ◽  
Mutant Strain ◽  
Legionella Pneumophila ◽  
Regulatory Networks ◽  
Acanthamoeba Castellanii ◽  
Logarithmic Phase ◽  
Insertion Mutation ◽  
Wild Type ◽  
E Coli ◽  
Resistance To Stress

ABSTRACT To investigate regulatory networks in Legionella pneumophila, the gene encoding the homolog of theEscherichia coli stress and stationary-phase sigma factor RpoS was identified by complementation of an E. coli rpoSmutation. An open reading frame that is approximately 60% identical to the E. coli rpoS gene was identified. Western blot analysis showed that the level of L. pneumophila RpoS increased in stationary phase. An insertion mutation was constructed in therpoS gene on the chromosome of L. pneumophila, and the ability of this mutant strain to survive various stress conditions was assayed and compared with results for the wild-type strain. Both the mutant and wild-type strains were more resistant to stress when in stationary phase than when in the logarithmic phase of growth. This finding indicates that L. pneumophila RpoS is not required for a stationary-phase-dependent resistance to stress. Although the mutant strain was able to kill HL-60- and THP-1-derived macrophages, it could not replicate within a protozoan host,Acanthamoeba castellanii. These data suggest that L. pneumophila possesses a growth phase-dependent resistance to stress that is independent of RpoS control and that RpoS likely regulates genes that enable it to survive in the environment within protozoa. Our data indicate that the role of rpoS inL. pneumophila is very different from what has previously been reported for E. coli rpoS.

scholarly journals Phosphate transporter PstSCAB of Campylobacter jejuni is essential for lactate-dependent growth and colonization in chickens

2019 ◽  
Author(s):  
Ritam Sinha ◽  
Rhiannon M. LeVeque ◽  
Marvin Q. Bowlin ◽  
Michael J. Gray ◽  
Victor J. DiRita
Keyword(s):  
Stationary Phase ◽  
Campylobacter Jejuni ◽  
Phosphate Transporter ◽  
Multi Drug Resistance ◽  
Wild Type ◽  
Gastrointestinal Infections ◽  
Poor Growth ◽  
High Affinity ◽  
Polyphosphate Metabolism

AbstractCampylobacter jejuni causes acute gastroenteritis world-wide and is transmitted primarily through poultry, in which it is often a commensal member of the intestinal microbiota. Previous RNASeq experiments showed that transcripts from an operon encoding a high affinity phosphate transporter (PstSCAB) of C. jejuni were among the most abundant when grown in chickens. Elevated levels of the pstSCAB mRNA were also identified in an RNASeq experiment from human infection studies. In this study, we explore the role of PstSCAB in the biology and colonization potential of C. jejuni. Our experimental results demonstrate that cells lacking PstSCAB survive poorly in stationary phase, nutrient-limiting media, and under osmotic conditions reflective of those in the chicken. Polyphosphate levels in the mutant cells were elevated at stationary phase, consistent with alterations in expression of polyphosphate metabolism genes. C. jejuni were highly attenuated in colonization of newly hatched chicks, recovered at levels several orders of magnitude below wild type. Mutant and wild type grew similarly in complex media but the pstSCAB mutant exhibited a significant growth defect in minimal media supplemented with L-lactate, postulated as a carbon source in vivo. Poor growth in lactate correlated with diminished expression of acetogenesis pathway genes previously demonstrated as important for colonizing chickens. The phosphate transport system is thus essential for diverse aspects of C. jejuni physiology and in vivo fitness and survival.ImportanceC. jejuni causes millions of gastrointestinal infections annually worldwide. Poultry and poultry products are major sources of C. jejuni infection to human as the microbe is a commensal colonizer of the chicken gastrointestinal tract. Due to the emergence of multi-drug resistance in C. jejuni, there is need to identify alternative ways to control this pathogen. Genes encoding the high-affinity phosphate transporter PstSCAB were highly expressed during colonization of C. jejuni in chicken and human. In this study, we address the role this high-affinity phosphate transporter PstSCAB of C. jejuni on chicken colonization and for its general physiology. PstSCAB is required for colonization in chicken, metabolism and survival under different stress responses and during growth on lactate, a potential substrate for growth of C. jejuni in chickens. Our study highlights that PstSCAB may be an effective target to develop mechanisms to control the bacterial burden in both chicken and human.

scholarly journals Phosphate Transporter PstSCAB of Campylobacter jejuni Is a Critical Determinant of Lactate-Dependent Growth and Colonization in Chickens

2020 ◽  
Vol 202 (7) ◽  
Author(s):  
Ritam Sinha ◽  
Rhiannon M. LeVeque ◽  
Marvin Q. Bowlin ◽  
Michael J. Gray ◽  
Victor J. DiRita
Keyword(s):  
Stationary Phase ◽  
Campylobacter Jejuni ◽  
Phosphate Transporter ◽  
Rna Seq ◽  
Wild Type ◽  
Content Type ◽  
High Affinity ◽  
Polyphosphate Metabolism

ABSTRACT Campylobacter jejuni causes acute gastroenteritis worldwide and is transmitted primarily through poultry, in which it is often a commensal member of the intestinal microbiota. Previous transcriptome sequencing (RNA-Seq) experiment showed that transcripts from an operon encoding a high-affinity phosphate transporter (PstSCAB) of C. jejuni were among the most abundant when the bacterium was grown in chickens. Elevated levels of the pstSCAB mRNA were also identified in an RNA-Seq experiment from human infection studies. In this study, we explore the role of PstSCAB in the biology and colonization potential of C. jejuni. Our results demonstrate that cells lacking PstSCAB survive poorly in stationary phase, in nutrient-limiting media, and under osmotic conditions reflective of those in the chicken. Polyphosphate levels in the mutant cells were elevated at stationary phase, consistent with alterations in expression of polyphosphate metabolism genes. The mutant strain was highly attenuated for colonization of newly hatched chicks, with levels of bacteria at several orders of magnitude below wild-type levels. Mutant and wild type grew similarly in complex media, but the pstS::kan mutant exhibited a significant growth defect in minimal medium supplemented with l-lactate, postulated as a carbon source in vivo. Poor growth in lactate correlated with diminished expression of acetogenesis pathway genes previously demonstrated as important for colonizing chickens. The phosphate transport system is thus essential for diverse aspects of C. jejuni physiology and in vivo fitness and survival. IMPORTANCE Campylobacter jejuni causes millions of human gastrointestinal infections annually, with poultry a major source of infection. Due to the emergence of multidrug resistance in C. jejuni, there is need to identify alternative ways to control this pathogen. Genes encoding the high-affinity phosphate transporter PstSCAB are highly expressed by C. jejuni in chickens and humans. In this study, we address the role of PstSCAB on chicken colonization and other C. jejuni phenotypes. PstSCAB is required for colonization in chicken, metabolism and survival under different stress responses, and during growth on lactate, a potential growth substrate in chickens. Our study highlights that PstSCAB may be an effective target to develop mechanisms for controlling bacterial burden in both chicken and human.

scholarly journals Intracellular Growth of Legionella pneumophila Gives Rise to a Differentiated Form Dissimilar to Stationary-Phase Forms

2002 ◽  
Vol 70 (11) ◽  
pp. 6273-6283 ◽  
Author(s):  
Rafael A. Garduño ◽  
Elizabeth Garduño ◽  
Margot Hiltz ◽  
Paul S. Hoffman
Keyword(s):  
Stationary Phase ◽  
Hela Cells ◽  
Legionella Pneumophila ◽  
Protein A ◽  
Plaque Assay ◽  
Growth Cycle ◽  
Potential Marker ◽  
Legionnaires Disease

ABSTRACT When Legionella pneumophila grows in HeLa cells, it alternates between a replicative form and a morphologically distinct “cyst-like” form termed MIF (mature intracellular form). MIFs are also formed in natural amoebic hosts and to a lesser extent in macrophages, but they do not develop in vitro. Since MIFs accumulate at the end of each growth cycle, we investigated the possibility that they are in vivo equivalents of stationary-phase (SP) bacteria, which are enriched for virulence traits. By electron microscopy, MIFs appeared as short, stubby rods with an electron-dense, laminar outer membrane layer and a cytoplasm largely occupied by inclusions of poly-β-hydroxybutyrate and laminations of internal membranes originating from the cytoplasmic membrane. These features may be responsible for the bright red appearance of MIFs by light microscopy following staining with the phenolic Giménez stain. In contrast, SP bacteria appeared as dull red rods after Giménez staining and displayed a typical gram-negative cell wall ultrastructure. Outer membranes from MIFs and SP bacteria were equivalent in terms of the content of the peptidoglycan-bound and disulfide bond cross-linked OmpS porin, although additional proteins, including Hsp60 (which acts as an invasin for HeLa cells), were detected only in preparations from MIFs. Proteomic analysis revealed differences between MIFs and SP forms; in particular, MIFs were enriched for an ∼20-kDa protein, a potential marker of development. Compared with SP bacteria, MIFs were 10-fold more infectious by plaque assay, displayed increased resistance to rifampin (3- to 5-fold) and gentamicin (10- to 1,000-fold), resisted detergent-mediated lysis, and tolerated high pH. Finally, MIFs had a very low respiration rate, consistent with a decreased metabolic activity. Collectively, these results suggest that intracellular L. pneumophila differentiates into a cyst-like, environmentally resilient, highly infectious, post-SP form that is distinct from in vitro SP bacteria. Therefore, MIFs may represent the transmissible environmental forms associated with Legionnaires' disease.

Role of Cu,Zn-SOD in the synthesis of endogenous vasodilator hydrogen peroxide during reactive hyperemia in mouse mesenteric microcirculation in vivo

2008 ◽  
Vol 294 (1) ◽  
pp. H441-H448 ◽  
Author(s):  
Toyotaka Yada ◽  
Hiroaki Shimokawa ◽  
Keiko Morikawa ◽  
Aya Takaki ◽  
Yoshiro Shinozaki ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Reactive Hyperemia ◽  
Fluorescent Microscopy ◽  
Artery Occlusion ◽  
Wild Type ◽  
Charge Coupled Device ◽  
In The Wild ◽  
Mesenteric Microcirculation

We have recently demonstrated that endothelium-derived hydrogen peroxide (H2O2) is an endothelium-derived hyperpolarizing factor and that endothelial Cu/Zn-superoxide dismutase (SOD) plays an important role in the synthesis of endogenous H2O2 in both animals and humans. We examined whether SOD plays a role in the synthesis of endogenous H2O2 during in vivo reactive hyperemia (RH), an important regulatory mechanism. Mesenteric arterioles from wild-type and Cu,Zn-SOD−/− mice were continuously observed by a pencil-type charge-coupled device (CCD) intravital microscope during RH (reperfusion after 20 and 60 s of mesenteric artery occlusion) in the cyclooxygenase blockade under the following four conditions: control, catalase alone, NG-monomethyl-l-arginine (l-NMMA) alone, and l-NMMA + catalase. Vasodilatation during RH was significantly decreased by catalase or l-NMMA alone and was almost completely inhibited by l-NMMA + catalase in wild-type mice, whereas it was inhibited by l-NMMA and l-NMMA + catalase in the Cu,Zn-SOD−/− mice. RH-induced increase in blood flow after l-NMMA was significantly increased in the wild-type mice, whereas it was significantly reduced in the Cu,Zn-SOD−/− mice. In mesenteric arterioles of the Cu,Zn-SOD−/− mice, Tempol, an SOD mimetic, significantly increased the ACh-induced vasodilatation, and the enhancing effect of Tempol was decreased by catalase. Vascular H2O2 production by fluorescent microscopy in mesenteric arterioles after RH was significantly increased in response to ACh in wild-type mice but markedly impaired in Cu,Zn-SOD−/− mice. Endothelial Cu,Zn-SOD plays an important role in the synthesis of endogenous H2O2 that contributes to RH in mouse mesenteric smaller arterioles.

scholarly journals Environmental Mimics and the Lvh Type IVA Secretion System Contribute to Virulence-Related Phenotypes of Legionella pneumophila

2006 ◽  
Vol 75 (2) ◽  
pp. 723-735 ◽  
Author(s):  
Purnima Bandyopadhyay ◽  
Shuqing Liu ◽  
Carolina B. Gabbai ◽  
Zeah Venitelli ◽  
Howard M. Steinman
Keyword(s):  
Stationary Phase ◽  
Legionella Pneumophila ◽  
Acanthamoeba Castellanii ◽  
Secretion System ◽  
Resistant Bacteria ◽  
Causative Organism ◽  
Legionnaires Disease ◽  
Intracellular Multiplication ◽  
Type Ivb Secretion ◽  
Type Ivb Secretion System

ABSTRACT Legionella pneumophila, the causative organism of Legionnaires' disease, is a fresh-water bacterium and intracellular parasite of amoebae. This study examined the effects of incubation in water and amoeba encystment on L. pneumophila strain JR32 and null mutants in dot/icm genes encoding a type IVB secretion system required for entry, delayed acidification of L. pneumophila-containing phagosomes, and intracellular multiplication when stationary-phase bacteria infect amoebae and macrophages. Following incubation of stationary-phase cultures in water, mutants in dotA and dotB, essential for function of the type IVB secretion system, exhibited entry and delay of phagosome acidification comparable to that of strain JR32. Following encystment in Acanthamoeba castellanii and reversion of cysts to amoeba trophozoites, dotA and dotB mutants exhibited intracellular multiplication in amoebae. The L. pneumophila Lvh locus, encoding a type IVA secretion system homologous to that in Agrobacterium tumefaciens, was required for restoration of entry and intracellular multiplication in dot/icm mutants following incubation in water and amoeba encystment and was required for delay of phagosome acidification in strain JR32. These data support a model in which the Dot/Icm type IVB secretion system is conditionally rather than absolutely required for L. pneumophila virulence-related phenotypes. The data suggest that the Lvh type IVA secretion system, previously thought to be dispensable, is involved in virulence-related phenotypes under conditions mimicking the spread of Legionnaires' disease from environmental niches. Since environmental amoebae are implicated as reservoirs for an increasing number of environmental pathogens and for drug-resistant bacteria, the environmental mimics developed here may be useful in virulence studies of other pathogens.

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