scholarly journals Recycling of the Anhydro-N-Acetylmuramic Acid Derived from Cell Wall Murein Involves a Two-Step Conversion to N-Acetylglucosamine-Phosphate

2005 ◽  
Vol 187 (11) ◽  
pp. 3643-3649 ◽  
Author(s):  
Tsuyoshi Uehara ◽  
Kyoko Suefuji ◽  
Noelia Valbuena ◽  
Brian Meehan ◽  
Michael Donegan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Cell Wall ◽  
Deletion Mutant ◽  
Biosynthetic Pathway ◽  
Reverse Genetics ◽  
Side Wall ◽  
Cell Extract ◽  
Amino Sugars ◽  
Kinase Gene

ABSTRACT Escherichia coli breaks down over 60% of the murein of its side wall and reuses the component amino acids to synthesize about 25% of the cell wall for the next generation. The amino sugars of the murein are also efficiently recycled. Here we show that the 1,6-anhydro-N-acetylmuramic acid (anhMurNAc) is returned to the biosynthetic pathway by conversion to N-acetylglucosamine-phosphate (GlcNAc-P). The sugar is first phosphorylated by anhydro- N -acetylmuramic acid kinase (AnmK), yielding MurNAc-P, and this is followed by action of an etherase which cleaves the bond between d-lactic acid and the N-acetylglucosamine moiety of MurNAc-P, yielding GlcNAc-P. The kinase gene has been identified by a reverse genetics method. The enzyme was overexpressed, purified, and characterized. The cell extract of an anmK deletion mutant totally lacked activity on anhMurNAc. Surprisingly, in the anmK mutant, anhMurNAc did not accumulate in the cytoplasm but instead was found in the medium, indicating that there was rapid efflux of free anhMurNAc.

scholarly journals The N-Acetyl-d-Glucosamine Kinase of Escherichia coli and Its Role in Murein Recycling

2004 ◽  
Vol 186 (21) ◽  
pp. 7273-7279 ◽  
Author(s):  
Tsuyoshi Uehara ◽  
James T. Park
Keyword(s):  
Escherichia Coli ◽  
Deletion Mutant ◽  
Alternative Pathway ◽  
Side Wall ◽  
Cell Extract ◽  
Diaminopimelic Acid ◽  
Terminal Sequence ◽  
E Coli ◽  
Cell Extracts ◽  
Kinase A

ABSTRACT N-Acetyl-d-glucosamine (GlcNAc) is a major component of bacterial cell wall murein and the lipopolysaccharide of the outer membrane. During growth, over 60% of the murein of the side wall is degraded, and the major products, GlcNAc-anhydro-N-acetylmuramyl peptides, are efficiently imported into the cytoplasm and cleaved to release GlcNAc, anhydro-N-acetylmuramic acid, murein tripeptide (l-Ala-d-Glu-meso-diaminopimelic acid), and d-alanine. Like murein tripeptide, GlcNAc is readily recycled, and this process was thought to involve phosphorylation, since GlcNAc-6-phosphate (GlcNAc-6-P) is efficiently used to synthesize murein or lipopolysaccharide or can be metabolized by glycolysis. Since the gene for GlcNAc kinase had not been identified, in this work we purified GlcNAc kinase (NagK) from Escherichia coli cell extracts and identified the gene by determining the N-terminal sequence of the purified kinase. A nagK deletion mutant lacked phosphorylated GlcNAc in its cytoplasm, and the cell extract of the mutant did not phosphorylate GlcNAc, indicating that NagK is the only GlcNAc kinase expressed in E. coli. Unexpectedly, GlcNAc did not accumulate in a nagK nagEBACD mutant, though both GlcNAc and GlcNAc-6-P accumulate in the nagEBACD mutant, suggesting the existence of an alternative pathway (presumably repressed by GlcNAc-6-P) that reutilizes GlcNAc without the involvement of NagK.

Cotranslational folding of nascent proteins on Escherichia coli ribosomes

1995 ◽  
Vol 73 (11-12) ◽  
pp. 1199-1207 ◽  
Author(s):  
Boyd Hardesty ◽  
Wieslaw Kudlicki ◽  
O. W. Odom ◽  
Tong Zhang ◽  
Diane McCarthy ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Synthetic Peptide ◽  
Deletion Mutant ◽  
Full Length ◽  
Release Factor ◽  
Terminal Sequence ◽  
Ribosomal Subunits ◽  
Chaperone Protein ◽  
Cotranslational Folding

Evidence is presented for cotranslational folding of rhodanese or ricin during its synthesis on Escherichia coli ribosomes. During transcription–translation, full-length but enzymatically inactive polypeptides accumulated as peptidyl-tRNA on the ribosomes. These polypeptides were activated and released by subsequent incubation with the bacterial chaperones and with release factor (RF-2). Coumarin was incorporated cotranslationally at the N-terminus of the nascent protein from fluorophore-S-Ac-Met-tRNAf. Changes in fluorescence indicated that DnaJ bound to the nascent proteins and to a fluorescently labeled synthetic peptide corresponding to the N-terminal 17 amino acids of bovine rhodanese. This peptide also bound to 70S ribosomes or 50S subunits but not to 30S subunits. It inhibited activation and RF-2-dependent release of the full-length ribosome-bound rhodanese. A deletion mutant of rhodanese lacking the N-terminal 23 amino acids was not accumulated on the ribosome but was synthesized very efficiently. However, the protein that was formed was enzymatically inactive. DnaJ did not bind to this deletion mutant on ribosomes. We conclude that the chaperone-mediated reactions facilitate binding of the N-terminal sequence of nascent proteins to a specific site on 50S ribosomal subunits where it blocks release. The ribosome-bound protein undergoes chaperone-mediated reactions that are required for folding into an enzymatically active conformation.Key words: protein synthesis, ribosome, chaperone, protein folding, nascent peptide.

scholarly journals Identification and Characterization of an Archaeon-Specific Riboflavin Kinase

2008 ◽  
Vol 190 (7) ◽  
pp. 2615-2618 ◽  
Author(s):  
Zahra Mashhadi ◽  
Hong Zhang ◽  
Huimin Xu ◽  
Robert H. White
Keyword(s):  
Escherichia Coli ◽  
Metal Ion ◽  
Biosynthetic Pathway ◽  
Recombinant Expression ◽  
Cell Extract ◽  
Flavin Mononucleotide ◽  
E Coli ◽  
Gene Recombinant ◽  
Identification And Characterization

ABSTRACT The riboflavin kinase in Methanocaldococcus jannaschii has been identified as the product of the MJ0056 gene. Recombinant expression of the MJ0056 gene in Escherichia coli led to a large increase in the amount of flavin mononucleotide (FMN) in the E. coli cell extract. The unexpected features of the purified recombinant enzyme were its use of CTP as the phosphoryl donor and the absence of a requirement for added metal ion to catalyze the formation of FMN. Identification of this riboflavin kinase fills another gap in the archaeal flavin biosynthetic pathway. Some divalent metals were found to be potent inhibitors of the reaction. The enzyme represents a unique CTP-dependent family of kinases.

Cell-Wall Amino-Acids and Amino-Sugars

Nature ◽  
1957 ◽  
Vol 180 (4581) ◽  
pp. 338-339 ◽  
Author(s):  
M. R. J. SALTON
Keyword(s):  
Amino Acids ◽  
Cell Wall ◽  
Amino Sugars

Relationship between Bdellovibrio bacteriovorus 6-5-S and autoclaved host bacteria

1972 ◽  
Vol 18 (12) ◽  
pp. 1941-1948 ◽  
Author(s):  
S. F. Crothers ◽  
H. B. Fackrell ◽  
J. C. C. Huang ◽  
J. Robinson
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Growth Rate ◽  
Reducing Sugars ◽  
Culture Fluid ◽  
Amino Sugars ◽  
Buffer Solution ◽  
Bdellovibrio Bacteriovorus ◽  
Initial Concentration ◽  
E Coli

Bdellovibrio bacteriovorus, strain 6-5-S, multiplied in the presence of washed suspensions of Escherichia coli, Spirillum serpens VHL, and Bacillus megaterium which had been autoclaved for 5 min at 121C. No intracellular life cycle was observed. Bdellovibrio bacteriovorus was also able to multiply in an extract from autoclaved E. coli cells after the cells had been removed by centrifugation. Growth of the parasite was dependent upon the addition of Ca2+ and Mg2+ to the buffer solution. The growth rate and yield of B. bacteriovorus on autoclaved cells were not affected by the initial concentration of the parasite. During multiplication of B. bacteriovorus, amino acids, amino sugars, and reducing sugars were released into the culture fluid.

Haemophilus vaginalis 594, a Gram-negative organism?

1971 ◽  
Vol 17 (7) ◽  
pp. 865-869 ◽  
Author(s):  
B. Sue Criswell ◽  
Judith H. Marston ◽  
Wayne A. Stenback ◽  
S. H. Black ◽  
Herman L. Gardner
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Cell Wall ◽  
Bacillus Megaterium ◽  
Teichoic Acid ◽  
Biochemical Properties ◽  
Positive Bacterium ◽  
Gram Negative ◽  
Bacterium Bacillus ◽  
Escherichia Coli B

The fine structure of Haemophilus vaginalis 594 (ATCC 14018) was examined by electron microscopy, and the biochemical composition of its cell wall was determined. For comparison, similar studies were done with a Gram-positive bacterium, Bacillus megaterium KM, and a Gram-negative bacterium, Escherichia coli B. Both Haemophilus vaginalis 594 and Escherichia coli B possessed a multiple-layered cell wall containing 11 to 14 amino acids, a low mucopeptide content, and no teichoic acid. In contrast, Bacillus megaterium KM had a thick, amorphous cell wall with five amino acids, high mucopeptide content, and detectable amounts of teichoic acid. Haemophilus vaginalis 594 resembled Escherichia coli, a member of the Gram-negative group of organisms. The structural and biochemical properties of Haemophilus vaginalis, which are described in detail, may prove useful in determining the ultimate taxonomic position of this species.

scholarly journals Identification of MpaA, an Amidase in Escherichia coli That Hydrolyzes the γ-d-Glutamyl-meso-Diaminopimelate Bond in Murein Peptides

2003 ◽  
Vol 185 (2) ◽  
pp. 679-682 ◽  
Author(s):  
Tsuyoshi Uehara ◽  
James T. Park
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Double Mutant ◽  
Biosynthetic Pathway ◽  
Bacillus Sphaericus ◽  
Cell Extract ◽  
Diaminopimelic Acid ◽  
Multicopy Plasmid ◽  
Amino Acid Sequence Homology ◽  
Hydrolysis Of

ABSTRACT MpaA amidase was identified in Escherichia coli by its amino acid sequence homology with the ENP1 endopeptidase from Bacillus sphaericus. The enzymatic activity of MpaA, i.e., hydrolysis of the γ-d-glutamyl-diaminopimelic acid bond in the murein tripeptide l-alanyl-γ-d-glutamyl-meso-diaminopimelic acid, was demonstrated in the cell extract of a strain expressing mpaA from a multicopy plasmid. An mpaA mpl (murein peptide ligase) double mutant accumulated large amounts of murein tripeptide in its cytoplasm, consistent with the premise that MpaA degrades the tripeptide if its recycling via the peptidoglycan biosynthetic pathway is blocked.

scholarly journals Role of DLP12 lysis genes in Escherichia coli biofilm formation

Microbiology ◽  
2011 ◽  
Vol 157 (6) ◽  
pp. 1640-1650 ◽  
Author(s):  
Faustino A. Toba ◽  
Mitchell G. Thompson ◽  
Bryan R. Campbell ◽  
Lauren M. Junker ◽  
Karl-Gustav Rueggeberg ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Biofilm Formation ◽  
Wall Stress ◽  
Wild Type ◽  
Lambda Phage ◽  
Kinase Gene ◽  
Abiotic Surfaces ◽  
K 12 ◽  
Biofilm Development

Phages have recently been implicated as important in biofilm development, although the mechanisms whereby phages impact biofilms remain unclear. One defective lambdoid phage carried by Escherichia coli K-12 is DLP12. Among the genes found in DLP12 are essD, ybcS and rzpD/rzoD, which are homologues of the Lambda phage genes encoding cell-lysis proteins (S, R and Rz/Rz1). The role that these DLP12 lysis genes play in biofilm formation was examined in deletion mutants of E. coli PHL628, a curli-overproducing, biofilm-forming K-12 derivative. Strains lacking essD, ybcS and rzpD/rzoD were unable to form wild-type biofilms. While all mutants were compromised in attachment to abiotic surfaces and aggregated less well than the wild-type, the effect of the essD knockout on biofilm formation was less dramatic than that of deleting ybcS or rzpD/rzoD. These results were consistent with electron micrographs of the mutants, which showed a decreased number of curli fibres on cell surfaces. Also consistent with this finding, we observed that expression from the promoter of csgB, which encodes the curli subunits, was downregulated in the mutants. As curli production is transcriptionally downregulated in response to cell wall stress, we challenged the mutants with SDS and found them to be more sensitive to the detergent than the wild-type. We also examined the release of 14C-labelled peptidoglycan from the mutants and found that they did not lose labelled peptidoglycan to the same extent as the wild-type. Given that curli production is known to be suppressed by N-acetylglucosamine 6-phosphate (NAG-6P), a metabolite produced during peptidoglycan recycling, we deleted nagK, the N-acetylglucosamine kinase gene, from the lysis mutants and found that this restored curli production. This suggested that deletion of the lysis genes affected cell wall status, which was transduced to the curli operon by NAG-6P via an as yet unknown mechanism. These observations provide evidence that the S, R and Rz/Rz1 gene homologues encoded by DLP12 are not merely genetic junk, but rather play an important, though undefined, role in cell wall maintenance.

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