scholarly journals Regulation of Hypercompetence in Legionella pneumophila

2004 ◽  
Vol 186 (12) ◽  
pp. 3814-3825 ◽  
Author(s):  
Jessica A. Sexton ◽  
Joseph P. Vogel
Keyword(s):  
Legionella Pneumophila ◽  
Bacterial Species ◽  
Single Species ◽  
Great Promise ◽  
Dna Uptake ◽  
Bacterial Strains ◽  
Aerobic Growth ◽  
Laboratory Conditions ◽  
Normal Laboratory

ABSTRACT Although many bacteria are known to be naturally competent for DNA uptake, this ability varies dramatically between species and even within a single species, some isolates display high levels of competence while others seem to be completely nontransformable. Surprisingly, many nontransformable bacterial strains appear to encode components necessary for DNA uptake. We believe that many such strains are actually competent but that this ability has been overlooked because standard laboratory conditions are inappropriate for competence induction. For example, most strains of the gram-negative bacterium Legionella pneumophila are not competent under normal laboratory conditions of aerobic growth at 37°C. However, it was previously reported that microaerophilic growth at 37°C allows L. pneumophila serogroup 1 strain AA100 to be naturally transformed. Here we report that another L. pneumophila serogroup 1 strain, Lp02, can also be transformed under these conditions. Moreover, Lp02 can be induced to high levels of competence by a second set of conditions, aerobic growth at 30°C. In contrast to Lp02, AA100 is only minimally transformable at 30°C, indicating that Lp02 is hypercompetent under these conditions. To identify potential causes of hypercompetence, we isolated mutants of AA100 that exhibited enhanced DNA uptake. Characterization of these mutants revealed two genes, proQ and comR, that are involved in regulating competence in L. pneumophila. This approach, involving the isolation of hypercompetent mutants, shows great promise as a method for identifying natural transformation in bacterial species previously thought to be nontransformable.

scholarly journals A multiplex CRISPR interference tool for virulence gene interrogation in Legionella pneumophila

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Nicole A. Ellis ◽  
Byoungkwan Kim ◽  
Jessica Tung ◽  
Matthias P. Machner
Keyword(s):  
Legionella Pneumophila ◽  
Bacterial Species ◽  
Virulence Gene ◽  
Polymerase Activity ◽  
Growth Defect ◽  
Great Promise ◽  
Macrophage Infection ◽  
Crispr Interference ◽  
Crispr Array ◽  
Virulence Protein

AbstractCatalytically inactive dCas9 imposes transcriptional gene repression by sterically precluding RNA polymerase activity at a given gene to which it was directed by CRISPR (cr)RNAs. This gene silencing technology, known as CRISPR interference (CRISPRi), has been employed in various bacterial species to interrogate genes, mostly individually or in pairs. Here, we developed a multiplex CRISPRi platform in the pathogen Legionella pneumophila capable of silencing up to ten genes simultaneously. Constraints on precursor-crRNA expression were overcome by combining a strong promoter with a boxA element upstream of a CRISPR array. Using crRNAs directed against virulence protein-encoding genes, we demonstrated that CRISPRi is fully functional not only during growth in axenic media, but also during macrophage infection, and that gene depletion by CRISPRi recapitulated the growth defect of deletion strains. By altering the position of crRNA-encoding spacers within the CRISPR array, our platform achieved the gradual depletion of targets that was mirrored by the severity in phenotypes. Multiplex CRISPRi thus holds great promise for probing large sets of genes in bulk in order to decipher virulence strategies of L. pneumophila and other bacterial pathogens.

scholarly journals Silencing of natural transformation by an RNA chaperone and a multitarget small RNA

2016 ◽  
Vol 113 (31) ◽  
pp. 8813-8818 ◽  
Author(s):  
Laetitia Attaiech ◽  
Aïda Boughammoura ◽  
Céline Brochier-Armanet ◽  
Omran Allatif ◽  
Flora Peillard-Fiorente ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Natural Transformation ◽  
Bacterial Species ◽  
Physiological State ◽  
Uptake System ◽  
Dna Uptake ◽  
Exogenous Dna ◽  
Major Mechanism ◽  
Transcriptional Gene Regulation ◽  
High Level

A highly conserved DNA uptake system allows many bacteria to actively import and integrate exogenous DNA. This process, called natural transformation, represents a major mechanism of horizontal gene transfer (HGT) involved in the acquisition of virulence and antibiotic resistance determinants. Despite evidence of HGT and the high level of conservation of the genes coding the DNA uptake system, most bacterial species appear non-transformable under laboratory conditions. In naturally transformable species, the DNA uptake system is only expressed when bacteria enter a physiological state called competence, which develops under specific conditions. Here, we investigated the mechanism that controls expression of the DNA uptake system in the human pathogenLegionella pneumophila. We found that a repressor of this system displays a conserved ProQ/FinO domain and interacts with a newly characterizedtrans-acting sRNA, RocR. Together, they target mRNAs of the genes coding the DNA uptake system to control natural transformation. This RNA-based silencing represents a previously unknown regulatory means to control this major mechanism of HGT. Importantly, these findings also show that chromosome-encoded ProQ/FinO domain-containing proteins can assisttrans-acting sRNAs and that this class of RNA chaperones could play key roles in post-transcriptional gene regulation throughout bacterial species.

scholarly journals A multiplex CRISPR interference tool for virulence gene interrogation in an intracellular pathogen

2020 ◽  
Author(s):  
Nicole A. Ellis ◽  
Byoungkwan Kim ◽  
Matthias P. Machner
Keyword(s):  
Legionella Pneumophila ◽  
Bacterial Species ◽  
Virulence Gene ◽  
Polymerase Activity ◽  
Growth Defect ◽  
Great Promise ◽  
Macrophage Infection ◽  
Large Sets ◽  
Virulence Protein ◽  
Rna Polymerase Activity

AbstractIn the absence of target cleavage, catalytically inactive dCas9 imposes transcriptional gene repression by sterically precluding RNA polymerase activity at a given gene to which it was directed by CRISPR (cr)RNAs. This gene silencing technology, referred to as CRISPR interference (CRISPRi), has been employed in various bacterial species to interrogate genes, mostly individually or in pairs. Here, we developed a multiplex CRISPRi platform in the pathogen Legionella pneumophila capable of silencing up to ten genes simultaneously. Constraints on precursor-crRNA expression by Rho-dependent transcription termination were overcome by combining a strong processive promoter with a boxA element upstream of a repeat/spacer array. Using crRNAs directed against virulence protein-encoding genes, we demonstrated that CRISPRi is fully functional not only during growth in axenic media, but also during macrophage infection, and that gene depletion by CRISPRi fully recapitulated the growth defect of deletion strains. Importantly, by altering the position of crRNA-encoding spacers within the repeat/spacer array, our platform achieved the gradual depletion of targets that was mirrored by the severity in phenotypes. Multiplex CRISPRi thus holds great promise for probing large sets of genes in bulk in order to decipher virulence strategies of L. pneumophila and other bacterial pathogens.

scholarly journals The Expanded Universe of Prokaryotic Argonaute Proteins

mBio ◽  
2018 ◽  
Vol 9 (6) ◽  
Author(s):  
Sergei Ryazansky ◽  
Andrey Kulbachinskiy ◽  
Alexei A. Aravin
Keyword(s):  
Rna Interference ◽  
Nucleic Acid ◽  
Genome Engineering ◽  
Bacterial Species ◽  
Host Cells ◽  
Bacterial Strains ◽  
Domains Of Life ◽  
Genomic Studies ◽  
Basic Biology

ABSTRACT Members of the ancient family of Argonaute (Ago) proteins are present in all domains of life. The common feature of Ago proteins is the ability to bind small nucleic acid guides and use them for sequence-specific recognition—and sometimes cleavage—of complementary targets. While eukaryotic Ago (eAgo) proteins are key players in RNA interference and related pathways, the properties and functions of these proteins in archaeal and bacterial species have just started to emerge. We undertook comprehensive exploration of prokaryotic Ago (pAgo) proteins in sequenced genomes and revealed their striking diversity in comparison with eAgos. Many pAgos contain divergent variants of the conserved domains involved in interactions with nucleic acids, while having extra domains that are absent in eAgos, suggesting that they might have unusual specificities in the nucleic acid recognition and cleavage. Many pAgos are associated with putative nucleases, helicases, and DNA binding proteins in the same gene or operon, suggesting that they are involved in target processing. The great variability of pAgos revealed by our analysis opens new ways for exploration of their functions in host cells and for their use as potential tools in genome editing. IMPORTANCE The eukaryotic Ago proteins and the RNA interference pathways they are involved in are widely used as a powerful tool in research and as potential therapeutics. In contrast, the properties and functions of prokaryotic Ago (pAgo) proteins have remained poorly understood. Understanding the diversity and functions of pAgos holds a huge potential for discovery of new cellular pathways and novel tools for genome manipulations. Only few pAgos have been characterized by structural or biochemical approaches, while previous genomic studies discovered about 300 proteins in archaeal and eubacterial genomes. Since that time the number of bacterial strains with sequenced genomes has greatly expanded, and many previously sequenced genomes have been revised. We undertook comprehensive analysis of pAgo proteins in sequenced genomes and almost tripled the number of known genes of this family. Our research thus forms a foundation for further experimental characterization of pAgo functions that will be important for understanding of the basic biology of these proteins and their adoption as a potential tool for genome engineering in the future.

scholarly journals Characterization of gtf, a Glucosyltransferase Gene in the Genomes of Pediococcus parvulus and Oenococcus oeni, Two Bacterial Species Commonly Found in Wine

2008 ◽  
Vol 74 (13) ◽  
pp. 4079-4090 ◽  
Author(s):  
Marguerite Dols-Lafargue ◽  
Hyo Young Lee ◽  
Claire Le Marrec ◽  
Alain Heyraud ◽  
Gérard Chambat ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Oenococcus Oeni ◽  
Mobile Elements ◽  
The Other ◽  
Bacterial Strains ◽  
Model Medium ◽  
Polysaccharide Synthesis

ABSTRACT “Ropiness” is a bacterial alteration in wines, beers, and ciders, caused by β-glucan-synthesizing pediococci. A single glucosyltransferase, Gtf, controls ropy polysaccharide synthesis. In this study, we show that the corresponding gtf gene is also present on the chromosomes of several strains of Oenococcus oeni isolated from nonropy wines. gtf is surrounded by mobile elements that may be implicated in its integration into the chromosome of O. oeni. gtf is expressed in all the gtf + strains, and β-glucan is detected in the majority of these strains. Part of this β-glucan accumulates around the cells forming a capsule, while the other part is liberated into the medium together with heteropolysaccharides. Most of the time, this polymer excretion does not lead to ropiness in a model medium. In addition, we show that wild or recombinant bacterial strains harboring a functional gtf gene (gtf +) are more resistant to several stresses occurring in wine (alcohol, pH, and SO2) and exhibit increased adhesion capacities compared to their gtf mutant variants.

scholarly journals Characterization of Phascolarctobacterium succinatutens sp. nov., an Asaccharolytic, Succinate-Utilizing Bacterium Isolated from Human Feces

2011 ◽  
Vol 78 (2) ◽  
pp. 511-518 ◽  
Author(s):  
Yohei Watanabe ◽  
Fumiko Nagai ◽  
Masami Morotomi
Keyword(s):  
Bacterial Species ◽  
Short Chain Fatty Acids ◽  
Pcr Analysis ◽  
Rrna Gene ◽  
Gi Tract ◽  
Bacterial Strains ◽  
High Sequence Identity ◽  
Healthy Human ◽  
Content Type

ABSTRACTIsolation, cultivation, and characterization of the intestinal microorganisms are important for understanding the comprehensive physiology of the human gastrointestinal (GI) tract microbiota. Here, we isolated two novel bacterial strains, YIT 12067Tand YIT 12068, from the feces of healthy human adults. Phylogenetic analysis indicated that they belonged to the same species and were most closely related toPhascolarctobacterium faeciumACM 3679T, with 91.4% to 91.5% 16S rRNA gene sequence similarities, respectively. Substrate availability tests revealed that the isolates used only succinate; they did not ferment any other short-chain fatty acids or carbohydrates tested. When these strains were cocultured with the xylan-utilizing and succinate-producing bacteriumParaprevotella xylaniphilaYIT 11841T, in medium supplemented with xylan but not succinate, their cell numbers became 2 to 3 orders of magnitude higher than those of the monoculture; succinate became undetectable, and propionate was formed. Database analysis revealed that over 200 uncultured bacterial clones from the feces of humans and other mammals showed high sequence identity (>98.7%) to YIT 12067T. Real-time PCR analysis also revealed that YIT 12067T-like bacteria were present in 21% of human fecal samples, at an average level of 3.34 × 108cells/g feces. These results indicate that YIT 12067T-like bacteria are distributed broadly in the GI tract as subdominant members that may adapt to the intestinal environment by specializing to utilize the succinate generated by other bacterial species. The phylogenetic and physiological properties of YIT 12067Tand YIT 12068 suggest that these strains represent a novel species, which we have designatedPhascolarctobacterium succinatutenssp. nov.

scholarly journals THE IDENTIFICATION, PRODUCTION AND CHARACTERIZATION OF NATTOKINASE, BACTERIOCIN FROM BACTERIAL SPECIES

2019 ◽  
Vol 2 (4) ◽  
pp. 91
Author(s):  
Lal Krishna
Keyword(s):  
Enzyme Activity ◽  
Bacillus Subtilis ◽  
Pathogenic Bacteria ◽  
Blood Clot ◽  
Bacterial Species ◽  
Antagonistic Activity ◽  
Bacterial Strains ◽  
Wide Range

The study was aimed at identification, production and characterization of nattokinase, bacteriocin from bacterial species. Nattokinase and bacteriocins finds a wide range of applications in Pharmaceutical industry, health care and medicine. Nattokinase is a highly active fibrinolytic enzyme secreted by Bacillus subtilis and bacteriocins are proteinaceous toxins produced by Lactobacillus to inhibit the growth of closely related bacterial strains. Bacillus subtilis and Lactobacillus isolates shown positive results to microscopic, biochemical analysis.  The nattokinase and bacteriocins were produced by optimizing the media. The enzymes were purified by ammonium sulfate precipitation and HPLC. The enzyme activity for nattokinase was found at 7 mg/ml, pH 8.0 and temperature 48 ºC and the enzyme activity for bacteriocin was found at 3.9 mg/ml, pH 6.5 and temperature 30 °C. Bacteriocins from Lactobacillus showed good antagonistic activity against pathogenic bacteria. Nattokinase from Bacillus subtilis played a significant role in thrombolytic and anti-coagulation at in vitro. The results indicated that the pure enzyme has a potential in dissolving blood clot.

scholarly journals Characterization of Two Unique Cold-Active Lipases Derived from a Novel Deep-Sea Cold Seep Bacterium

2021 ◽  
Vol 9 (4) ◽  
pp. 802
Author(s):  
Chenchen Guo ◽  
Rikuan Zheng ◽  
Ruining Cai ◽  
Chaomin Sun ◽  
Shimei Wu
Keyword(s):  
Deep Sea ◽  
Bacterial Species ◽  
Deep Ocean ◽  
Cold Seep ◽  
Bacterial Strains ◽  
E Coli ◽  
Cold Active ◽  
Genes Encoding ◽  
Potential Inhibitors

The deep ocean microbiota has unexplored potential to provide enzymes with unique characteristics. In order to obtain cold-active lipases, bacterial strains isolated from the sediment of the deep-sea cold seep were screened, and a novel strain gcc21 exhibited a high lipase catalytic activity, even at the low temperature of 4 °C. The strain gcc21 was identified and proposed to represent a new species of Pseudomonas according to its physiological, biochemical, and genomic characteristics; it was named Pseudomonas marinensis. Two novel encoding genes for cold-active lipases (Lipase 1 and Lipase 2) were identified in the genome of strain gcc21. Genes encoding Lipase 1 and Lipase 2 were respectively cloned and overexpressed in E. coli cells, and corresponding lipases were further purified and characterized. Both Lipase 1 and Lipase 2 showed an optimal catalytic temperature at 4 °C, which is much lower than those of most reported cold-active lipases, but the activity and stability of Lipase 2 were much higher than those of Lipase 1 under different tested pHs and temperatures. In addition, Lipase 2 was more stable than Lipase 1 when treated with different metal ions, detergents, potential inhibitors, and organic solvents. In a combination of mutation and activity assays, catalytic triads of Ser, Asp, and His in Lipase 1 and Lipase 2 were demonstrated to be essential for maintaining enzyme activity. Phylogenetic analysis showed that both Lipase 1 and Lipase 2 belonged to lipase family III. Overall, our results indicate that deep-sea cold seep is a rich source for novel bacterial species that produce potentially unique cold-active enzymes.

scholarly journals Towards Understanding the Molecular Basis of Nitric Oxide-Regulated Group Behaviors in Pathogenic Bacteria

2018 ◽  
Vol 11 (3) ◽  
pp. 205-215 ◽  
Author(s):  
Dominique E. Williams ◽  
Elizabeth M. Boon
Keyword(s):  
Nitric Oxide ◽  
Legionella Pneumophila ◽  
Pathogenic Bacteria ◽  
Bacterial Species ◽  
Planktonic Cell ◽  
Antibiotic Resistant ◽  
Abiotic Surfaces ◽  
Biofilm Dispersal ◽  
Identification And Characterization

Pathogenic bacteria have many strategies for causing disease in humans. One such strategy is the ability to live both as single-celled motile organisms or as part of a community of bacteria called a biofilm. Biofilms are frequently adhered to biotic or abiotic surfaces and are extremely antibiotic resistant. Upon biofilm dispersal, bacteria become more antibiotic susceptible but are also able to readily infect another host. Various studies have shown that low, nontoxic levels of nitric oxide (NO) may induce biofilm dispersal in many bacterial species. While the molecular details of this phenotype remain largely unknown, in several species, NO has been implicated in biofilm-to-planktonic cell transitions via ligation to 1 of 2 characterized NO sensors, NosP or H-NOX. Based on the data available to date, it appears that NO binding to H-NOX or NosP triggers a downstream response based on changes in cellular cyclic di-GMP concentrations and/or the modulation of quorum sensing. In order to develop applications for control of biofilm infections, the identification and characterization of biofilm dispersal mechanisms is vital. This review focuses on the efforts made to understand NO-mediated control of H-NOX and NosP pathways in the 3 pathogenic bacteria Legionella pneumophila, Vibrio cholerae, and Pseudomonas aeruginosa.

scholarly journals Characterization of six clinical isolates of Chimaeribacter gen. nov., a novel genus related to the Yersiniaceae family and the three species Chimaeribacter arupi sp. nov., Chimaeribacter coloradensis sp. nov, and Chimaeribacter californicus sp. nov.

2020 ◽  
Vol 70 (4) ◽  
pp. 2703-2712 ◽  
Author(s):  
Alessandro Rossi ◽  
Mark A. Fisher
Keyword(s):  
Type Strain ◽  
Single Species ◽  
Individual Species ◽  
Rrna Gene ◽  
Bacterial Strains ◽  
Gram Negative ◽  
Novel Genus ◽  
Content Type ◽  
Link Type

Eight genetically related, Gram-negative bacterial strains, isolated from clinical specimens between 2012 and 2016, were submitted to arup Laboratories for species identification. The lack of species- or genus-level matches in curated 16S rRNA gene databases prompted us to undertake the polyphasic characterization of these so far undescribed organisms. Six isolates available for additional testing were oxidase negative, catalase positive, pleomorphic, Gram-negative rods displaying temperature-dependent motility and producing yellow-pigmented colonies with three distinct morphotypes: medium-sized shiny, large mucoid and agar-pitting. Biochemical reactions and sugar fermentation patterns were most similar to members of the genus Serratia . Fatty acid profiles were highly similar across all six organisms, with the major components being: C16 : 0; C17 : 0 cyclo; C14 : 0 3-OH/iso-C16 : 1 I; C18 : 1 ω7c; and C16 : 1 ω7c/C16 : 1 ω6c. Whole-genome comparisons and multi locus sequence analysis (using the coding genes atpD, rpoB, gyrB and infB) suggest that the strains here described constitute three individual species within a novel genus related to the family Yersiniaceae . We propose for this novel taxon the name Chimaeribacter gen. nov., referring to the presentation of multiple characteristics typical of distinct Enterobacterales genera within a single organism. Four isolates are representative of a single species: Chimaeribacter arupi sp. nov (2016-Iso1, 2016-Iso2, type strain 2016-Iso3T=DSM 110101T=ATCC TSD-180T and 2013-Iso5). The remaining two isolates constitute the novel species Chimaeribacter coloradensis sp. nov. (type strain 2016-Iso4T=DSM 110102T=ATCC TSD-182T) and Chimaeribacter californicus sp. nov. (type strain 2015-Iso6T=DSM 110100T=ATCC TSD-181T). Our work provides the first formal characterization of the genus Chimaeribacter and forms the basis to study its taxonomic diversity.

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