scholarly journals Bacillus subtilis YhaM, a Member of a New Family of 3′-to-5′ Exonucleases in Gram-Positive Bacteria

2002 ◽  
Vol 184 (22) ◽  
pp. 6250-6259 ◽  
Author(s):  
Irina A. Oussenko ◽  
Roberto Sanchez ◽  
David H. Bechhofer
Keyword(s):  
Bacillus Subtilis ◽  
Mrna Turnover ◽  
Wild Type ◽  
Rnase R ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
New Family ◽  
Double Stranded Dna ◽  
Ob Fold ◽  
Related Proteins

ABSTRACT A strain of Bacillus subtilis lacking two 3′-to-5′ exoribonucleases, polynucleotide phosphorylase (PNPase) and RNase R, was used to purify another 3′-to-5′ exoribonuclease, which is encoded by the yhaM gene. YhaM was active in the presence of Mn2+ (or Co2+), was inactive in the presence of Mg2+, and could also degrade single-stranded DNA. The half-life of bulk mRNA in a mutant lacking PNPase, RNase R, and YhaM was not significantly different from that of the wild type, suggesting the existence of additional activities that can participate in mRNA turnover. Sequence homologues of YhaM were found only in gram-positive organisms. The Staphylococcus aureus homologue, CBF1, which had been characterized as a double-stranded DNA binding protein involved in plasmid replication, was also shown to be an Mn2+-dependent exoribonuclease. YhaM protein has a C-terminal “HD domain,” found in metal-dependent phosphohydrolases. By structure modeling, it was shown that YhaM also contains an N-terminal “OB-fold,” present in many oligosaccharide- and oligonucleotide-binding proteins. The combination of these two domains is unique. Thus, YhaM and 10 related proteins from gram-positive organisms constitute a new exonuclease family.

scholarly journals In Vitro Characterization of the Bacillus subtilis Protein Tyrosine Phosphatase YwqE

2005 ◽  
Vol 187 (10) ◽  
pp. 3384-3390 ◽  
Author(s):  
Ivan Mijakovic ◽  
Lucia Musumeci ◽  
Lutz Tautz ◽  
Dina Petranovic ◽  
Robert A. Edwards ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Tyrosine Phosphatase ◽  
Protein Tyrosine Phosphatases ◽  
Class Ii ◽  
Gram Positive ◽  
Protein Tyrosine ◽  
Gram Positive Bacteria ◽  
New Family ◽  
In Vitro Characterization

ABSTRACT Both gram-negative and gram-positive bacteria possess protein tyrosine phosphatases (PTPs) with a catalytic Cys residue. In addition, many gram-positive bacteria have acquired a new family of PTPs, whose first characterized member was CpsB from Streptococcus pneumoniae. Bacillus subtilis contains one such CpsB-like PTP, YwqE, in addition to two class II Cys-based PTPs, YwlE and YfkJ. The substrates for both YwlE and YfkJ are presently unknown, while YwqE was shown to dephosphorylate two phosphotyrosine-containing proteins implicated in UDP-glucuronate biosynthesis, YwqD and YwqF. In this study, we characterize YwqE, compare the activities of the three B. subtilis PTPs (YwqE, YwlE, and YfkJ), and demonstrate that the two B. subtilis class II PTPs do not dephosphorylate the physiological substrates of YwqE.

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 phgABC, a Three-Gene Operon Required for Growth of Streptococcus pneumoniae in Hyperosmotic Medium and In Vivo

2004 ◽  
Vol 72 (8) ◽  
pp. 4579-4588 ◽  
Author(s):  
Jeremy S. Brown ◽  
Sarah M. Gilliland ◽  
Shilpa Basavanna ◽  
David W. Holden
Keyword(s):  
Streptococcus Pneumoniae ◽  
Mutant Strain ◽  
Compatible Solutes ◽  
Wild Type ◽  
Gram Positive Bacteria ◽  
Mutant Strains ◽  
Increased Sensitivity ◽  
Related Proteins ◽  
And Oxidative Stress

ABSTRACT To cause disease, bacterial pathogens need to be able to adapt to the physiological conditions found within the host, including an osmolality of approximately 290 mosmol kg−1. While investigating Streptococcus pneumoniae genes contained within pneumococcal pathogenicity island 1, we identified a three-gene operon of unknown function termed phgABC. PhgC has a domain with similarity to diacylglycerol kinases of eukaryotes and is the first described member of a family of related proteins found in many gram-positive bacteria. phgA and phgC mutant strains were constructed by insertional duplication mutagenesis and found to have impaired growth under conditions of high osmotic and oxidative stress. The compatible solutes proline and glycine betaine improved growth of the wild-type and the phgA mutant strains in hyperosmolar medium, and when analyzed by electron microscopy, the cellular morphology of the phgA mutant strain was unaffected by osmotic stress. The phgA and phgC mutant strains were reduced in virulence in models of both systemic and pulmonary infection. As the virulence of the phgA mutant strain was not restored in gp91phox−/− mice and the phgA and phgC mutant strains had reduced growth in both blood and serum, the reduced virulence of these strains is unlikely to be due to increased sensitivity to the respiratory burst of phagocytes but is, instead, due to impaired growth at physiological osmolality.

scholarly journals A Comparative Genome Analysis Identifies Distinct Sorting Pathways in Gram-Positive Bacteria

2004 ◽  
Vol 72 (5) ◽  
pp. 2710-2722 ◽  
Author(s):  
David Comfort ◽  
Robert T. Clubb
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Surface Proteins ◽  
Sequence Motifs ◽  
Comparative Genome Analysis ◽  
Gram Positive ◽  
Microbial Genomes ◽  
Searchable Database ◽  
Gram Positive Bacteria ◽  
Related Proteins

ABSTRACT Surface proteins in gram-positive bacteria are frequently required for virulence, and many are attached to the cell wall by sortase enzymes. Bacteria frequently encode more than one sortase enzyme and an even larger number of potential sortase substrates that possess an LPXTG-type cell wall sorting signal. In order to elucidate the sorting pathways present in gram-positive bacteria, we performed a comparative analysis of 72 sequenced microbial genomes. We show that sortase enzymes can be partitioned into five distinct subfamilies based upon their primary sequences and that most of their substrates can be predicted by making a few conservative assumptions. Most bacteria encode sortases from two or more subfamilies, which are predicted to function nonredundantly in sorting proteins to the cell surface. Only ∼20% of sortase-related proteins are most closely related to the well-characterized Staphylococcus aureus SrtA protein, but nonetheless, these proteins are responsible for anchoring the majority of surface proteins in gram-positive bacteria. In contrast, most sortase-like proteins are predicted to play a more specialized role, with each anchoring far fewer proteins that contain unusual sequence motifs. The functional sortase-substrate linkage predictions are available online (http://www.doe-mbi.ucla.edu/Services/Sortase/ ) in a searchable database.

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 A Conserved Class II Type Thioester Domain-Containing Adhesin Is Required for Efficient Conjugation in Bacillus subtilis

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
César Gago-Córdoba ◽  
Jorge Val-Calvo ◽  
David Abia ◽  
Alberto Díaz-Talavera ◽  
Andrés Miguel-Arribas ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Bacillus Subtilis ◽  
Recipient Cell ◽  
Class Ii ◽  
Pair Formation ◽  
Conjugative Plasmid ◽  
Mating Pair ◽  
Gram Positive ◽  
Content Type ◽  
Gram Positive Bacteria

ABSTRACT Conjugation, the process by which a DNA element is transferred from a donor to a recipient cell, is the main horizontal gene transfer route responsible for the spread of antibiotic resistance and virulence genes. Contact between a donor and a recipient cell is a prerequisite for conjugation, because conjugative DNA is transferred into the recipient via a channel connecting the two cells. Conjugative elements encode proteins dedicated to facilitating the recognition and attachment to recipient cells, also known as mating pair formation. A subgroup of the conjugative elements is able to mediate efficient conjugation during planktonic growth, and mechanisms facilitating mating pair formation will be particularly important in these cases. Conjugative elements of Gram-negative bacteria encode conjugative pili, also known as sex pili, some of which are retractile. Far less is known about mechanisms that promote mating pair formation in Gram-positive bacteria. The conjugative plasmid pLS20 of the Gram-positive bacterium Bacillus subtilis allows efficient conjugation in liquid medium. Here, we report the identification of an adhesin gene in the pLS20 conjugation operon. The N-terminal region of the adhesin contains a class II type thioester domain (TED) that is essential for efficient conjugation, particularly in liquid medium. We show that TED-containing adhesins are widely conserved in Gram-positive bacteria, including pathogens where they often play crucial roles in pathogenesis. Our study is the first to demonstrate the involvement of a class II type TED-containing adhesin in conjugation. IMPORTANCE Bacterial resistance to antibiotics has become a serious health care problem. The spread of antibiotic resistance genes between bacteria of the same or different species is often mediated by a process named conjugation, where a donor cell transfers DNA to a recipient cell through a connecting channel. The first step in conjugation is recognition and attachment of the donor to a recipient cell. Little is known about this first step, particularly in Gram-positive bacteria. Here, we show that the conjugative plasmid pLS20 of Bacillus subtilis encodes an adhesin protein that is essential for effective conjugation. This adhesin protein has a structural organization similar to adhesins produced by other Gram-positive bacteria, including major pathogens, where the adhesins serve in attachment to host tissues during colonization and infection. Our findings may thus also open novel avenues to design drugs that inhibit the spread of antibiotic resistance by blocking the first recipient-attachment step in conjugation.

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 Apparent involvement of a multidrug transporter in the fluoroquinolone resistance of Streptococcus pneumoniae.

1997 ◽  
Vol 41 (6) ◽  
pp. 1396-1398 ◽  
Author(s):  
N N Baranova ◽  
A A Neyfakh
Keyword(s):  
Streptococcus Pneumoniae ◽  
Ethidium Bromide ◽  
Fluoroquinolone Resistance ◽  
Cross Resistance ◽  
Multidrug Transporter ◽  
Wild Type ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Energy Dependent

A Streptococcus pneumoniae strain selected for resistance to ethidium bromide demonstrated enhanced energy-dependent efflux of this toxic dye. Both the ethidium resistance and the ethidium efflux could be inhibited by the plant alkaloid reserpine. The ethidium-selected cells demonstrated cross-resistance to the fluoroquinolones norfloxacin and ciprofloxacin; this resistance could also be completely reversed by reserpine. Furthermore, reserpine potentiated the susceptibility of wild-type S. pneumoniae to fluoroquinolones and ethidium. The most plausible explanation for these results is that S. pneumoniae, like some other gram-positive bacteria, expresses a reserpine-sensitive multidrug transporter, which may play an important role in both intrinsic and acquired resistances of this pathogen to fluoroquinolone therapy.

Sign in / Sign up

Export Citation Format

Share Document