scholarly journals Tn551-mediated insertional inactivation of the fmtB gene encoding a cell wall-associated protein abolishes methicillin resistance in Staphylococcus aureus

2000 ◽  
Vol 45 (4) ◽  
pp. 421-431 ◽  
Author(s):  
H. Komatsuzawa ◽  
K. Ohta ◽  
M. Sugai ◽  
T. Fujiwara ◽  
P. Glanzmann ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Methicillin Resistance ◽  
Gene Encoding ◽  
Insertional Inactivation ◽  
A Cell

scholarly journals Morphological and Genetic Differences in Two Isogenic Staphylococcus aureus Strains with Decreased Susceptibilities to Vancomycin

2003 ◽  
Vol 47 (2) ◽  
pp. 568-576 ◽  
Author(s):  
Andrea Reipert ◽  
Kerstin Ehlert ◽  
Thomas Kast ◽  
Gabriele Bierbaum
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Methicillin Resistance ◽  
Resistance Mechanism ◽  
Cross Linking ◽  
Spontaneous Mutant ◽  
Chromosomal Dna ◽  
False Target ◽  
A Cell ◽  
Wall Profile

ABSTRACT Many VISA (vancomycin intermediately resistant Staphylococcus aureus) strains are characterized by increased cell wall biosynthesis and decreased cross-linking of the peptide side chains, leading to accumulation of free d-alanyl-d-alanine termini in the peptidoglycan, which act as false target sites for vancomycin. A spontaneous mutant of methicillin-resistant VISA strain SA137/93A (vancomycin MIC [E-test], 8 μg/ml), called SA137/93G, showed increased resistance to vancomycin (MIC [E-test], 12 μg/ml). Analysis of the resistance profile of the mutant revealed a loss of β-lactam resistance with a concomitant increase in resistance to glycopeptides. In both strains, cell wall thickness was 1.4-fold greater than that of control isolates. However, cross-linking of the cell wall was drastically lower in SA137/93A than in SA137/93G. The sensitivity of strain SA137/93G to β-lactams was due to loss of the β-lactamase plasmid and a deletion that comprises 32.5 kb of the methicillin resistance cassette SCCmec, as well as 65.4 kb of chromosomal DNA. A spontaneous mutant of SA137/93G with higher sensitivity to vancomycin displayed a cell wall profile similar, in some respects, to that of an fmhB mutant. Results described here and elsewhere show that the only feature common to all VISA strains is a thickened cell wall, which may play a central role in the vancomycin resistance mechanism.

scholarly journals β-Lactam Resistance in Staphylococcus aureus Cells That Do Not Require a Cell Wall for Integrity

2005 ◽  
Vol 49 (12) ◽  
pp. 5075-5080 ◽  
Author(s):  
Elizabeth Fuller ◽  
Catherine Elmer ◽  
Fiona Nattress ◽  
Richard Ellis ◽  
Glenda Horne ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Methicillin Resistance ◽  
Intact Cell ◽  
Penicillin G ◽  
Coding Regions ◽  
Osmotic Stability ◽  
A Cell ◽  
Triton X ◽  
Resistant Cells

ABSTRACT Staphylococcus aureus ATCC 9144 cells with defective cell walls were generated on a medium with elevated osmolality in the presence of sublethal levels of penicillin G. On removal of antibiotic pressure, the cells exhibited stable penicillin and methicillin resistance. The resistance was homogeneous and its acquisition was enhanced following transient cell wall-defective growth. The resistant cells were mecA negative, β-lactamase negative and did not contain any mutations in the coding regions of pbp genes. When penicillin was added back to resistant cells, they continued to grow and produced a diffuse cell wall that was resistant to the action by lysostaphin but was very sensitive to lysis with Triton X-100. These data indicate that the resistant cells are not dependent upon an intact cell wall for osmotic stability and they are able to switch readily to this mode of growth in the presence of penicillin G.

scholarly journals Role of a Sodium-Dependent Symporter Homologue in the Thermosensitivity of β-Lactam Antibiotic Resistance and Cell Wall Composition in Staphylococcus aureus

2007 ◽  
Vol 52 (2) ◽  
pp. 505-512 ◽  
Author(s):  
Krzysztof Sieradzki ◽  
Marilyn Chung ◽  
Alexander Tomasz
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Antibiotic Resistance ◽  
Methicillin Resistance ◽  
Homology Search ◽  
Sodium Dependent ◽  
Lactam Antibiotic ◽  
A Cell ◽  
Mrsa Strains ◽  
High Level

ABSTRACT Expression of high-level β-lactam resistance is known to be thermosensitive in many methicillin-resistant Staphylococcus aureus (MRSA) strains, including strain COL, in which the high methicillin MIC for cultures grown at 37°C (800 μg/ml) was reduced to 12 μg/ml at 42°C. COL grew faster at 42°C than at 37°C and at the higher temperature produced cell walls of abnormal composition: there was an over-representation of the monomeric muropeptide without the oligoglycine chain and an increase in the representation of multimers that contained this wall component as the donor molecule. Screening of a Tn551 insertional library for mutants, in which the high and homogenous β-lactam antibiotic resistance of strain COL is retained at 42°C, identified mutant C245, which expressed high-level methicillin resistance and produced a cell wall of normal composition independent of the temperature. The Tn551 inactivated gene was found, by homology search, to encode for a sodium-dependent symporter, homologues of which are ubiquitous in both prokaryotic and eukaryotic genomes. Inactivation of this putative symporter in several heteroresistant clinical MRSA isolates caused striking increases in the level of their β-lactam resistance.

scholarly journals The AbcA Transporter of Staphylococcus aureus Affects Cell Autolysis

2001 ◽  
Vol 45 (2) ◽  
pp. 407-412 ◽  
Author(s):  
Gesine Schrader-Fischer ◽  
Brigitte Berger-Bächi
Keyword(s):  
Staphylococcus Aureus ◽  
Methicillin Resistance ◽  
Erythromycin Resistance ◽  
Gene Encoding ◽  
Transcription Analysis ◽  
Global Regulators ◽  
Cell Extracts ◽  
Insertional Inactivation ◽  
Cell Autolysis

ABSTRACT Increased production of penicillin-binding protein PBP 4 is known to increase peptidoglycan cross-linking and contributes to methicillin resistance in Staphylococcus aureus. The pbp4gene shares a 400-nucleotide intercistronic region with the divergently transcribed abcA gene, encoding an ATP-binding cassette transporter of unknown function. Our study revealed that methicillin stimulated abcA transcription but had no effects onpbp4 transcription. Analysis of abcA expression in mutants defective for global regulators showed that abcAis under the control of agr. Insertional inactivation ofabcA by an erythromycin resistance determinant did not influence pbp4 transcription, nor did it alter resistance to methicillin and other cell wall-directed antibiotics. However,abcA mutants showed spontaneous partial lysis on plates containing subinhibitory concentrations of methicillin due to increased spontaneous autolysis. Since the autolytic zymograms of cell extracts were identical in mutants and parental strains, we postulate an indirect role of AbcA in control of autolytic activities and in protection of the cells against methicillin.

scholarly journals Role of murE in the Expression of β-Lactam Antibiotic Resistance in Staphylococcus aureus

2004 ◽  
Vol 186 (6) ◽  
pp. 1705-1713 ◽  
Author(s):  
S. Gardete ◽  
A. M. Ludovice ◽  
R. G. Sobral ◽  
S. R. Filipe ◽  
H. de Lencastre ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Methicillin Resistance ◽  
Inducible Promoter ◽  
Northern Analysis ◽  
Penicillin Binding Protein ◽  
Drastic Reduction ◽  
Precursor Pool ◽  
Lactam Antibiotic

ABSTRACT It was shown earlier that Tn551 inserted into the C-terminal region of murE of parental methicillin-resistant Staphylococcus aureus strain COL causes a drastic reduction in methicillin resistance, accompanied by accumulation of UDP-MurNAc dipeptide in the cell wall precursor pool and incorporation of these abnormal muropeptides into the peptidoglycan of the mutant. Methicillin resistance was recovered in a suppressor mutant. The murE gene of the same strain was then put under the control of the isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible promoter P spac . Bacteria grown in the presence of suboptimal concentrations of IPTG accumulated UDP-MurNAc dipeptide in the cell wall precursor pool. Both growth rates and methicillin resistance levels (but not resistance to other antibiotics) were a function of the IPTG concentration. Northern analysis showed a gradual increase in the transcription of murE and also in the transcription of pbpB and mecA, parallel with the increasing concentrations of IPTG in the medium. A similar increase in the transcription of pbpB and mecA, the structural genes of penicillin-binding protein 2 (PBP2) and PBP2A, was also detected in the suppressor mutant. The expression of these two proteins, which are known to play critical roles in the mechanism of staphylococcal methicillin resistance, appears to be—directly or indirectly—under the control of the murE gene. Our data suggest that the drastic reduction of the methicillin MIC seen in the murE mutant may be caused by the insufficient cellular amounts of these two PBPs.

How penicillin kills bacteria: progress and problems

1971 ◽  
Vol 179 (1057) ◽  
pp. 369-383 ◽  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Bacterial Cell ◽  
Crosslinking Reaction ◽  
Free System ◽  
Whole Cells ◽  
Uridine Nucleotide ◽  
History Of ◽  
A Cell ◽  
Specific Inhibitors

Penicillins and cephalosporins are specific inhibitors of the biosynthesis of bacterial cell walls. This discovery was first made in 1957 and was based on two observations. First, penicillins induced the formation of protoplasts or spheroplasts in bacteria (organisms in which the cell wall has been lost or weakened) (Lederberg 1957). Secondly, a uridine nucleotide accumulated in Staphylococcus aureus and other bacteria inhibited by penicillin which had a striking relationship to the composition of the cell wall (Park & Strominger 1957). It was therefore suggested that this nucleotide was an activated precursor of the wall. Over the next decade, a great deal of work was carried out in order to elucidate the structure of the bacterial cell wall and the mechanism of its biosynthesis from the uridine nucleotides and other precursors (reviewed by Strominger 1970; Strominger & Ghuysen 1967; Ghuysen 1968). It was demonstrated that interpeptide cross-links were an important structural feature of the wall. Several kinds of experiments carried out with whole cells indicated that the final step in cell wall synthesis, the crosslinking reaction catalysed by a transpeptidase, was the site of action of penicillin (Wise & Park 1965; Tipper & Strominger 1965 a , b , 1968). Finally, in 1966, the transpeptidase catalysing this cross-linking reaction was obtained in a cell-free system and shown to be a penicillin-sensitive enzyme (Izaki, Matsuhashi & Strominger 1966, 1968). The history of these developments has been reviewed elsewhere (Strominger 1970), and in the present paper, attention will be focused on recent studies of the penicillin-sensitive transpeptidase and other penicillinsensitive activities found in bacterial cell membranes. First, however, it is necessary to describe briefly the structure of the cell wall of bacteria and the nature of the inhibited reactions. The walls of bacteria consist of glycan strands in which two sugars, acetylglucosamine (X) and acetylmuramic acid (Y), strictly alternate (figure 1). Four such glycan strands are represented in figure 1. The acetylmuramic acid residues of the polymer are substituted by a tetrapeptide (represented in the figure by open circles). The peptidoglycan strand (i.e., the glycan substituted by the tetrapeptide) are cross-linked to one another by means of an interpeptide bridge which is to some extent a genus-specific character­istic. In the genus Staphylococcus aureus , the interpeptide bridge is a pentaglycine chain (represented in figure 1 by the closed circles) which extends from the carboxyl group on the terminal D-alanine residue of the tetrapeptide to the ∊-amino group of lysine, the third amino acid in the tetrapeptide chain. The wall of S . aureus is a very tightly knit structure in that virtually every peptide subunit is cross-linked to another subunit by means of this interpeptide bridge. Penicillins and cephalosporins are specific inhibitors of the reaction in which the cross-link is actually formed. This step is the last reaction in wall synthesis.

scholarly journals Tissue-Specific Expression of a Gene Encoding a Cell Wall-Localized Lipid Transfer Protein from Arabidopsis

1994 ◽  
Vol 105 (1) ◽  
pp. 35-45 ◽  
Author(s):  
S. Thoma ◽  
U. Hecht ◽  
A. Kippers ◽  
J. Botella ◽  
S. De Vries ◽  
...  
Keyword(s):  
Cell Wall ◽  
Lipid Transfer Protein ◽  
Lipid Transfer ◽  
Gene Encoding ◽  
Specific Expression ◽  
Tissue Specific ◽  
Tissue Specific Expression ◽  
Transfer Protein ◽  
A Cell

scholarly journals Fungal α-arabinofuranosidases of glycosyl hydrolase families 51 and 54 show a dual arabinofuranosyl- and galactofuranosyl-hydrolyzing activity

2012 ◽  
Vol 393 (8) ◽  
pp. 767-775 ◽  
Author(s):  
Boris Tefsen ◽  
Ellen L. Lagendijk ◽  
Joohae Park ◽  
Michiel Akeroyd ◽  
Doreen Schachtschabel ◽  
...  
Keyword(s):  
Cell Wall ◽  
Enzyme Activity ◽  
Molecular Modeling ◽  
Aspergillus Niger ◽  
Glycosyl Hydrolase ◽  
Hydrolase Activity ◽  
Gene Encoding ◽  
A Cell ◽  
Or Gene

Abstract Aspergillus niger possesses a galactofuranosidase activity, however, the corresponding enzyme or gene encoding this enzyme has never been identified. As evidence is mounting that enzymes exist with affinity for both arabinofuranose and galactofuranose, we investigated the possibility that α-l-arabinofuranosidases, encoded by the abfA and abfB genes, are responsible for the galactofuranosidase activity of A. niger. Characterization of the recombinant AbfA and AbfB proteins revealed that both enzymes do not only hydrolyze p-nitrophenyl-α-l-arabinofuranoside (pNp-α-Araf) but are also capable of hydrolyzing p-nitrophenyl-β-d-galactofuranoside (pNp-β-Galf). Molecular modeling of the AbfB protein with pNp-β-Galf confirmed the possibility for AbfB to interact with this substrate, similarly as with pNp-α-Araf. We also show that galactomannan, a cell wall compound of A. niger, containing β-linked terminal and internal galactofuranosyl moieties, can be degraded by an enzyme activity that is present in the supernatant of inulin-grown A. niger. Interestingly, purified AbfA and AbfB did not show this hydrolyzing activity toward A. nigergalactomannan. In summary, our studies demonstrate that AbfA and AbfB, α-l-arabinofuranosidases from different families, both contain a galactofuranose (Galf)-hydrolyzing activity. In addition, our data support the presence of a Galf-hydrolase activity expressed by A. niger that is capable of degrading fungal galactomannan.

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