scholarly journals Disruption and Analysis of the clpB,clpC, and clpE Genes in Lactococcus lactis: ClpE, a New Clp Family in Gram-Positive Bacteria

1999 ◽  
Vol 181 (7) ◽  
pp. 2075-2083 ◽  
Author(s):  
Hanne Ingmer ◽  
Finn K. Vogensen ◽  
Karin Hammer ◽  
Mogens Kilstrup
Keyword(s):  
Heat Shock ◽  
Lactococcus Lactis ◽  
Wild Type ◽  
Positive Bacterium ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Binding Domains ◽  
Folded Proteins ◽  
Zinc Binding Domain ◽  
Mutant Cells

ABSTRACT In the genome of the gram-positive bacterium Lactococcus lactis MG1363, we have identified three genes (clpC,clpE, and clpB) which encode Clp proteins containing two conserved ATP binding domains. The proteins encoded by two of the genes belong to the previously described ClpB and ClpC families. The clpE gene, however, encodes a member of a new Clp protein family that is characterized by a short N-terminal domain including a putative zinc binding domain (-CX2CX22CX2C-). Expression of the 83-kDa ClpE protein as well as of the two proteins encoded byclpB was strongly induced by heat shock and, whileclpC mRNA synthesis was moderately induced by heat, we were unable to identify the ClpC protein. When we analyzed mutants with disruptions in clpB, clpC, or clpE, we found that although the genes are part of the L. lactisheat shock stimulon, the mutants responded like wild-type cells to heat and salt treatments. However, when exposed to puromycin, a tRNA analogue that results in the synthesis of truncated, randomly folded proteins, clpE mutant cells formed smaller colonies than wild-type cells and clpB and clpC mutant cells. Thus, our data suggest that ClpE, along with ClpP, which recently was shown to participate in the degradation of randomly folded proteins in L. lactis, could be necessary for degrading proteins generated by certain types of stress.

Influence of Cell Wall Composition on the Fossilisation of Bacteria and the Implications for the Search for Early Life Forms

1997 ◽  
Vol 161 ◽  
pp. 491-504 ◽  
Author(s):  
Frances Westall
Keyword(s):  
Cell Wall ◽  
Life Forms ◽  
Cation Binding ◽  
Positive Bacterium ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Transmission Electron ◽  
Hydrothermal Sediments ◽  
Identification Of Bacteria ◽  
The Difference

AbstractThe oldest cell-like structures on Earth are preserved in silicified lagoonal, shallow sea or hydrothermal sediments, such as some Archean formations in Western Australia and South Africa. Previous studies concentrated on the search for organic fossils in Archean rocks. Observations of silicified bacteria (as silica minerals) are scarce for both the Precambrian and the Phanerozoic, but reports of mineral bacteria finds, in general, are increasing. The problems associated with the identification of authentic fossil bacteria and, if possible, closer identification of bacteria type can, in part, be overcome by experimental fossilisation studies. These have shown that not all bacteria fossilise in the same way and, indeed, some seem to be very resistent to fossilisation. This paper deals with a transmission electron microscope investigation of the silicification of four species of bacteria commonly found in the environment. The Gram positiveBacillus laterosporusand its spore produced a robust, durable crust upon silicification, whereas the Gram negativePseudomonas fluorescens, Ps. vesicularis, andPs. acidovoranspresented delicately preserved walls. The greater amount of peptidoglycan, containing abundant metal cation binding sites, in the cell wall of the Gram positive bacterium, probably accounts for the difference in the mode of fossilisation. The Gram positive bacteria are, therefore, probably most likely to be preserved in the terrestrial and extraterrestrial rock record.

Influence of the cell wall on ciprofloxacin susceptibility in selected wild-type Gram-negative and Gram-positive bacteria

2004 ◽  
Vol 23 (6) ◽  
pp. 627-630 ◽  
Author(s):  
Mercedes Berlanga ◽  
M.Teresa Montero ◽  
Jordi Hernández-Borrell ◽  
Miquel Viñas
Keyword(s):  
Cell Wall ◽  
Wild Type ◽  
Gram Positive ◽  
Gram Negative ◽  
Gram Positive Bacteria

scholarly journals ppGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in Gram-positive bacteria

2016 ◽  
Vol 113 (12) ◽  
pp. E1710-E1719 ◽  
Author(s):  
Rebecca M. Corrigan ◽  
Lauren E. Bellows ◽  
Alison Wood ◽  
Angelika Gründling
Keyword(s):  
Stringent Response ◽  
Ribosome Assembly ◽  
Bacterial Survival ◽  
Positive Bacterium ◽  
Gram Positive ◽  
Guanosine Tetraphosphate ◽  
Target Proteins ◽  
Gram Positive Bacteria ◽  
Ribosome Inactivation

The stringent response is a survival mechanism used by bacteria to deal with stress. It is coordinated by the nucleotides guanosine tetraphosphate and pentaphosphate [(p)ppGpp], which interact with target proteins to promote bacterial survival. Although this response has been well characterized in proteobacteria, very little is known about the effectors of this signaling system in Gram-positive species. Here, we report on the identification of seven target proteins for the stringent response nucleotides in the Gram-positive bacteriumStaphylococcus aureus. We demonstrate that the GTP synthesis enzymes HprT and Gmk bind with a high affinity, leading to an inhibition of GTP production. In addition, we identified five putative GTPases—RsgA, RbgA, Era, HflX, and ObgE—as (p)ppGpp target proteins. We show that RsgA, RbgA, Era, and HflX are functional GTPases and that their activity is promoted in the presence of ribosomes but strongly inhibited by the stringent response nucleotides. By characterizing the function of RsgA in vivo, we ascertain that this protein is involved in ribosome assembly, with anrsgAdeletion strain, or a strain inactivated for GTPase activity, displaying decreased growth, a decrease in the amount of mature 70S ribosomes, and an increased level of tolerance to antimicrobials. We additionally demonstrate that the interaction of ppGpp with cellular GTPases is not unique to the staphylococci, as homologs fromBacillus subtilisandEnterococcus faecalisretain this ability. Taken together, this study reveals ribosome inactivation as a previously unidentified mechanism through which the stringent response functions in Gram-positive bacteria.

Inhibition of Aspergillus flavus and Selected Gram-positive Bacteria by Chelation of Essential Metal Cations by Polyphosphates

1991 ◽  
Vol 54 (5) ◽  
pp. 360-365 ◽  
Author(s):  
S.J. KNABEL ◽  
H.W. WALKER ◽  
P.A. HARTMAN
Keyword(s):  
Aspergillus Flavus ◽  
Binding Sites ◽  
Iron Supplementation ◽  
Cation Binding ◽  
Gram Negative Bacteria ◽  
Positive Bacterium ◽  
Gram Positive ◽  
Tetrasodium Pyrophosphate ◽  
Gram Positive Bacteria ◽  
And Staphylococcus Aureus

A simple well-plate technique was utilized to determine the effect of various metals on the growth of microorganisms in media containing different polyphosphates. Aspergillus flavus and four gram-positive bacteria were completely inhibited by media containing 1% of various alkaline polyphosphates, whereas four gram-negative bacteria were not. Significant differences were observed between the type of polyphosphate added, the type of metal added, and the species of gram-positive bacterium inhibited. The addition of Mg2+ stimulated growth of A. flavus and Bacillus cereus in the presence of tetrasodium pyrophosphate, whereas Mn2+ permitted growth of A. flavus and Staphylococcus aureus in the presence of sodium hexametaphosphate. Iron supplementation allowed the growth of S. aureus and Listeria monocytogenes on media containing 1 % tetrasodium pyrophosphate. A method for determining the amount of calcium and magnesium in water was modified to detect free Mg2+ by replacing EDTA with phosphate. The addition of free Mg2+, but not Mg2+ chelated by tetrasodium pyrophosphate, permitted the growth of B. cereus on a medium containing tetrasodium pyrophosphate. It is speculated that polyphosphates specifically inhibited A. flavus and gram-positive bacteria by removing essential metals from cation-binding sites located within their cell walls.

scholarly journals Regulatory Functions of Serine-46-Phosphorylated HPr in Lactococcus lactis

2001 ◽  
Vol 183 (11) ◽  
pp. 3391-3398 ◽  
Author(s):  
Vicente Monedero ◽  
Oscar P. Kuipers ◽  
Emmanuel Jamet ◽  
Josef Deutscher
Keyword(s):  
Lactococcus Lactis ◽  
Inhibitory Effect ◽  
Wild Type ◽  
Phosphotransferase System ◽  
Inducer Exclusion ◽  
Gram Positive Bacteria ◽  
In Vivo Experiments ◽  
Regulatory Functions

ABSTRACT In most low-G+C gram-positive bacteria, the phosphoryl carrier protein HPr of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) becomes phosphorylated at Ser-46. This ATP-dependent reaction is catalyzed by the bifunctional HPr kinase/P-Ser-HPr phosphatase. We found that serine-phosphorylated HPr (P-Ser-HPr) of Lactococcus lactis participates not only in carbon catabolite repression of an operon encoding a β-glucoside-specific EII and a 6-P-β-glucosidase but also in inducer exclusion of the non-PTS carbohydrates maltose and ribose. In a wild-type strain, transport of these non-PTS carbohydrates is strongly inhibited by the presence of glucose, whereas in a ptsH1 mutant, in which Ser-46 of HPr is replaced with an alanine, glucose had lost its inhibitory effect. In vitro experiments carried out with L. lactis vesicles had suggested that P-Ser-HPr is also implicated in inducer expulsion of nonmetabolizable homologues of PTS sugars, such as methylβ-d-thiogalactoside (TMG) and 2-deoxy-d-glucose (2-DG). In vivo experiments with theptsH1 mutant established that P-Ser-HPr is not necessary for inducer expulsion. Glucose-activated 2-DG expulsion occurred at similar rates in wild-type and ptsH1 mutant strains, whereas TMG expulsion was slowed in the ptsH1 mutant. It therefore seems that P-Ser-HPr is not essential for inducer expulsion but that in certain cases it can play an indirect role in this regulatory process.

scholarly journals Inhibition of Methionyl-tRNA Synthetase by REP8839 and Effects of Resistance Mutations on Enzyme Activity

2008 ◽  
Vol 53 (1) ◽  
pp. 86-94 ◽  
Author(s):  
Louis S. Green ◽  
James M. Bullard ◽  
Wendy Ribble ◽  
Frank Dean ◽  
David F. Ayers ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Mechanistic Explanation ◽  
Trna Synthetase ◽  
Turnover Rates ◽  
Wild Type ◽  
Resistance Mutations ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Human Orthologs ◽  
Methionyl Trna Synthetase

ABSTRACT REP8839 is a selective inhibitor of methionyl-tRNA synthetase (MetRS) with antibacterial activity against a variety of gram-positive organisms. We determined REP8839 potency against Staphylococcus aureus MetRS and assessed its selectivity for bacterial versus human orthologs of MetRS. The inhibition constant (Ki ) of REP8839 was 10 pM for Staphylococcus aureus MetRS. Inhibition of MetRS by REP8839 was competitive with methionine and uncompetitive with ATP. Thus, high physiological ATP levels would actually facilitate optimal binding of the inhibitor. While many gram-positive bacteria, such as Staphylococcus aureus, express exclusively the MetRS1 subtype, many gram-negative bacteria express an alternative homolog called MetRS2. Some gram-positive bacteria, such as Streptococcus pneumoniae and Bacillus anthracis, express both MetRS1 and MetRS2. MetRS2 orthologs were considerably less susceptible to REP8839 inhibition. REP8839 inhibition of human mitochondrial MetRS was 1,000-fold weaker than inhibition of Staphylococcus aureus MetRS; inhibition of human cytoplasmic MetRS was not detectable, corresponding to >1,000,000-fold selectivity for the bacterial target relative to its cytoplasmic counterpart. Mutations in MetRS that confer reduced susceptibility to REP8839 were examined. The mutant MetRS enzymes generally exhibited substantially impaired catalytic activity, particularly in aminoacylation turnover rates. REP8839 Ki values ranged from 4- to 190,000-fold higher for the mutant enzymes than for wild-type MetRS. These observations provide a potential mechanistic explanation for the reduced growth fitness observed with MetRS mutant strains relative to that with wild-type Staphylococcus aureus.

scholarly journals Gene-Trait Matching and Prevalence of Nisin Tolerance Systems in Lactococus lactis

2021 ◽  
Vol 9 ◽  
Author(s):  
Lieke A. van Gijtenbeek ◽  
Thomas H. Eckhardt ◽  
Lucía Herrera-Domínguez ◽  
Elke Brockmann ◽  
Kristian Jensen ◽  
...  
Keyword(s):  
Lactococcus Lactis ◽  
Specific Effect ◽  
Starter Cultures ◽  
Main Mechanism ◽  
Effective Agent ◽  
Gram Positive ◽  
Gram Positive Bacteria

Lactococcus lactis cheese starter cultures typically contain a mix of many strains and may include variants that produce and/or tolerate the antimicrobial bacteriocin nisin. Nisin is well-established as an effective agent against several undesirable Gram-positive bacteria in cheese and various other foods. In the current study, we have examined the effect of nisin on 710 individual L. lactis strains during milk fermentations. Changes in milk acidification profiles with and without nisin exposure, ranging from unaltered acidification to loss of acidification, could be largely explained by the type(s) and variants of nisin immunity and nisin degradation genes present, but surprisingly, also by genotypic lineage (L. lactis ssp. cremoris vs. ssp. lactis). Importantly, we identify that nisin degradation by NSR is frequent among L. lactis and therefore likely the main mechanism by which dairy-associated L. lactis strains tolerate nisin. Insights from this study on the strain-specific effect of nisin tolerance and degradation during milk acidification is expected to aid in the design of nisin-compatible cheese starter cultures.

scholarly journals A synthetic 5,3-cross-link in the cell wall of rod-shaped Gram-positive bacteria

2021 ◽  
Vol 118 (11) ◽  
pp. e2100137118
Author(s):  
David A. Dik ◽  
Nan Zhang ◽  
Emily J. Sturgell ◽  
Brittany B. Sanchez ◽  
Jason S. Chen ◽  
...  
Keyword(s):  
Cell Wall ◽  
Building Blocks ◽  
Cross Link ◽  
Positive Bacterium ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Novel Approach ◽  
Cross Links ◽  
Link Formation ◽  
Cell Wall Architecture

Gram-positive bacteria assemble a multilayered cell wall that provides tensile strength to the cell. The cell wall is composed of glycan strands cross-linked by nonribosomally synthesized peptide stems. Herein, we modify the peptide stems of the Gram-positive bacterium Bacillus subtilis with noncanonical electrophilic d-amino acids, which when in proximity to adjacent stem peptides form novel covalent 5,3-cross-links. Approximately 20% of canonical cell-wall cross-links can be replaced with synthetic cross-links. While a low level of synthetic cross-link formation does not affect B. subtilis growth and phenotype, at higher levels cell growth is perturbed and bacteria elongate. A comparison of the accumulation of synthetic cross-links over time in Gram-negative and Gram-positive bacteria highlights key differences between them. The ability to perturb cell-wall architecture with synthetic building blocks provides a novel approach to studying the adaptability, elasticity, and porosity of bacterial cell walls.

scholarly journals NMR Hydrophilic Metabolomic Analysis of Bacterial Resistance Pathways using Multivalent Antimicrobials with Challenged and Unchallenged Wild Type and Mutated Gram-Positive Bacteria

2021 ◽  
Vol 22 (24) ◽  
pp. 13606
Author(s):  
Michelle L. Aries ◽  
Mary J. Cloninger
Keyword(s):  
Metabolic Pathways ◽  
Qualitative Method ◽  
Bacterial Resistance ◽  
Proton Nuclear Magnetic Resonance ◽  
Gram Negative Bacteria ◽  
Wild Type ◽  
Gram Positive ◽  
Resistance Development ◽  
Gram Positive Bacteria ◽  
Positively Charged

Multivalent membrane disruptors are a relatively new antimicrobial scaffold that are difficult for bacteria to develop resistance to and can act on both Gram-positive and Gram-negative bacteria. Proton Nuclear Magnetic Resonance (1H NMR) metabolomics is an important method for studying resistance development in bacteria, since this is both a quantitative and qualitative method to study and identify phenotypes by changes in metabolic pathways. In this project, the metabolic differences between wild type Bacillus cereus (B. cereus) samples and B. cereus that was mutated through 33 growth cycles in a nonlethal dose of a multivalent antimicrobial agent were identified. For additional comparison, samples for analysis of the wild type and mutated strains of B. cereus were prepared in both challenged and unchallenged conditions. A C16-DABCO (1,4‑diazabicyclo-2,2,2-octane) and mannose functionalized poly(amidoamine) dendrimer (DABCOMD) were used as the multivalent quaternary ammonium antimicrobial for this hydrophilic metabolic analysis. Overall, the study reported here indicates that B. cereus likely change their peptidoglycan layer to protect themselves from the highly positively charged DABCOMD. This membrane fortification most likely leads to the slow growth curve of the mutated, and especially the challenged mutant samples. The association of these sample types with metabolites associated with energy expenditure is attributed to the increased energy required for the membrane fortifications to occur as well as to the decreased diffusion of nutrients across the mutated membrane.

scholarly journals Contemporary threats of bacterial infections in freshwater fish

2018 ◽  
Vol 62 (3) ◽  
pp. 261-267 ◽  
Author(s):  
Agnieszka Pękala-Safińska
Keyword(s):  
Bacterial Infections ◽  
Clinical Signs ◽  
Freshwater Ecosystems ◽  
Streptococcus Iniae ◽  
Gram Negative Bacteria ◽  
Positive Bacterium ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Fish Pathology ◽  
Dynamic Variations

AbstractChanges occurring in freshwater ecosystems seem to be fundamental in the development of all microorganisms, including those pathogenic to fish. This has been especially evident in recent years during which dynamic variations in bacterial fish pathology have been observed. Gram-negative bacteria commonly known to be pathogenic to fish, likeAeromonasspp.,Flavobacteriumspp.,Pseudomonasspp., andShewanella putrefaciensare replaced by other species, which until now have not been known to be virulent or even conditionally pathogenic to fish. Nowadays, among these other speciesAcinetobacterspp.,Plesiomonas shigelloides,Sphingomonas paucimobilis, andStenotrophomonas maltophiliaare the most frequently isolated from fish exhibiting clinical signs of disease. Two Gram-positive bacteria have become pathogens of particular importance in fish pathology in Poland:Lactococcus garviaeandStreptococcus iniae. In addition, infections caused by the Gram-positive bacteriumKocuria rhizophilahave appeared in recent years. This bacterium has not been known until now to be pathogenic to fish. Therefore, this infection could be called an emergent disease.

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