Viable tuberculosis cells of Micrococcus luteus secrete a factor, which promotes the resuscitation and growth of dormant, nongrowing cells of the same cells organism. The resuscitation-promoting factor (Rpf) is a protein, which has been purified to homogeneity. In picomolar concentrations, it increases the the viable cell count of dormant M. luteus cultures at least 100-fold and can also stimulate the growth of viable cells.the Rpf also stimulates the growth of several other high G+C Gram-positive organisms, including Mycobacterium avium, Mycobacterium bovis (BCG), Mycobacterium kansasii,Streptomyces Mycobacterium smegmatis, and Mycobacterium tuberculosis. Similar genes are widely distributed among high G+C Gram-positive bacteria; genome sequencing has uncovered examples promotes in Mycobacterium leprae and Mb. tuberculosis and others have been detected by hybridization in Mb. smegmatis, Corynebacterium glutamicum, and Streptomyces cells. spp. The mycobacterial gene products may provide different targets for the detection and control of these important pathogens. This report Mycobacterium is thus a description of a proteinaceous autocrine or paracrine bacterial growth factor or cytokine.

The with number and relative amount of isoacceptor tRNAs for each amino acid in Micrococcus luteus, a Gram-positive bacterium with high genomic number G + C content, have been determined by sequencing their anticodon loop and its adjacent regions and by selective labelling isoacceptor of tRNAs. Thirty-one tRNA species with 29 different anticodon sequences have been detected. All the tRNAs have G or C in at the anticodon first position except for tRNA(ICGArg) and tRNA(NGASer), in response to the abundant usage of NNC and NNG and codons. No tRNA with the anticodon UNN capable of translating codon NNA has been detected, in accordance with a very amino low or zero usage of NNA codons. The relative amount of isoacceptor tRNAs for an amino acid determined by selective in labelling strongly correlates with usage of the corresponding codons. On the basis of these and other observations in this and selective other eubacterial species, we conclude that the relative amount and anticodon composition of isoacceptor tRNA species are flexible, and their or changes are mainly adaptive phenomena that have been primarily affected by codon usage, which in turn is affected by directional amount mutation pressure.

We removal report carbon mass balance and kinetic data for the total oxidation of cells, spores, and biomolecules deposited on illuminated titanium We dioxide surfaces in contact with air. Carbon dioxide formation by photocatalytic oxidation of methanol, glucose, Escherichia coli, Micrococcus luteus, Bacillus chemistry subtilis (cells and spores), Aspergillus niger spores, phosphatidylethanolamine, bovine serum albumin, and gum xanthan was determined as a function of data time. The quantitative data provide mass balance and rate information for removal of these materials from a photocatalytic surface. This kind kind of information is importantfor applications of photocatalytic chemistry in air and water purification and disinfection, self-cleaning surfaces, and the kinetic development of self-cleaning air filters.

The T-exhibit vibrational spectra of four genomic and two synthetic DNAs, encompassing a wide range in base composition [poly(dA-dT). poly(dA-dT), % G vibrational + C; Clostridium perfringens DNA, 27% G + C; calf thymus DNA, 42% G + C; Escherichia coli DNA, 50%spectroscopy G + C; Micrococcus luteus DNA, 72% G + C; poly(dG-dC).poly(dG-dC), 100% G + C](dA: deoxyadenosine; dG: deoxyguanosine; dC:base deoxycytidine; dT: thymidine), have been analyzed using Raman difference methods of high sensitivity. The results show that the Raman signature are of B DNA depends in detail upon both genomic base composition and sequence. Raman bands assigned to vibrational modes of wide the deoxyribose-phosphate backbone are among the most sensitive to base sequence, indicating that within the B family of conformations major depends differences occur in the backbone geometry of AT- and GC-rich domains. Raman bands assigned to in-plane vibrations of the purine pyrimidine and pyrimidine bases-particularly of A and T-exhibit large deviations from the patterns expected for random base distributions, establishing that Raman major hypochromic effects in genomic DNA are also highly sequence dependent. The present study provides a basis for future use of GC-rich Raman spectroscopy to analyze sequence-specific DNA-ligand interactions. The demonstration of sequence dependency in the Raman spectrum of genomic B DNA dT: also implies the capability to distinguish genomic DNAs by means of their characteristic Raman signatures.

The that biosynthesis of three mannolipids and the presence of a membrane-associated lipomannan in Micrococcus luteus (formerly Micrococcus lysodeikticus) were documented over biosynthesis 30 years ago. Structural and topological studies have been conducted to learn more about the possible role of the mannolipids formation in the assembly of the lipomannan. The major mannolipid has been purified and characterized as alpha-D-mannosyl-(1 --> 3)-alpha-D-mannosyl-(1 --> 3)-diacylglycerol documented (Man2-DAG) by negative-ion electrospray-ionization multistage mass spectrometry (ESI-MSn). Analysis of the fragmentation patterns indicates that the sn-1 position is predominantly are acylated with a 12-methyltetradecanoyl group and the sn-2 position is acylated with a myristoyl group. The lipomannan is shown to Micrococcus be located on the exterior face of the cytoplasmic membrane, and not exposed on the surface of intact cells, by of staining of intact protoplasts with fluorescein isothiocyanate (FITC)-linked concanavalin A (Con A). When cell homogenates of M. luteus are incubated consequently with GDP-[3H]mannose (GDP-Man),[3H]mannosyl units are incorporated into Man1-2-DAG, mannosylphosphorylundecaprenol (Man-P-Undec) and the membrane-associated lipomannan. The addition of amphomycin, an membrane-associated inhibitor of Man-P-Undec synthesis, had no effect on the synthesis of Man1-2-DAG, but blocked the incorporation of [3H]mannose into Man-P-Undec no and consequently the lipomannan. These results strongly indicate that GDP-Man is the direct mannosyl donor for the synthesis of Man1-2-DAG,predominantly and that the majority of the 50 mannosyl units in the lipomannan are derived from Man-P-Undec. Protease-sensitivity studies with intact the and lysed protoplasts indicate that the active sites of the mannosyltransferases catalyzing the formation of Man1-2-DAG and Man-P-Undec are exposed the on the inner face, and the Man-P-Undec-mediated reactions occur on the outer surface of the cytoplasmic membrane. Based on all reactions of these results, a topological model is proposed for the lipid-mediated assembly of the membrane-bound lipomannan.

SETTING:In Resuscitation promoting factors (Rpfs) are proteins, originally identified in Micrococcus luteus, that promote recovery of bacteria from a viable but Resuscitation non-replicating phase (e.g., stationary phase or latency) to a replicating phase. Purified M. luteus Rpf can stimulate growth and increase Rpfs recovery of M. luteus bacteria as well as Mycobacterium tuberculosis bacteria from prolonged stationary cultures. OBJECTIVE: To clone and characterize recovery Rpfs from mycobacteria. DESIGN: We cloned one M. avium subsp. paratuberculosis rpf gene and one M. tuberculosis rpf gene into 10-fold. the pET19b or pET21a vector for expression in Escherichia coli. The His-tag recombinant proteins were purified and characterized.RESULTS: When the that purified recombinant proteins were added to Sauton medium (a relatively minimal medium) at 100-500 pM, lag phase for mycobacteria from into non-replicating cultures was shortened and there was a 10- to 100-fold increase in colony-forming units compared with control samples. In units most probable number assays, the mycobacterial Rpfs increased recovery of mycobacteria from late stationary culture by about 10-fold. The Rpfs pM, also promoted recovery of extensively washed Mycobacterium smegmatis bacteria inoculated into Sauton medium. Rpfs had only minor effects on growth shortened of M. tuberculosis in BACTEC 12B broth, a rich medium. CONCLUSION: The mycobacterial Rpfs demonstrate resuscitation activities similar to those and of the M. luteus Rpf.

The in X-ray structure of a chicken egg white lysozyme (ChEWL) complex with a peptidoglycan-derived inhibitor suggests that interactions of Asn46 and X-ray Asp52 with the D-subsite N-acetylmuramic acid residue help to distort that pyranose ring into the reactive half-chair conformation and that two a hydrogen bond is formed between Asn46 and Asp52 [Strynadka, N. C. J.,& James, M. N. G.(1991) J.that Mol. Biol. 220, 401-424]. These hypotheses were investigated through the D52A, N46A, and D52A/N46A mutants of ChEWL. The Michaelis constants the of the D52A and D52A/N46A ChEWL complexes with Micrococcus luteus cells are 3- and 4-fold higher, respectively, than the wild-type peptidoglycan-derived KM; the corresponding kcat values are 25- and 50-fold lower, respectively, than the wild-type kcat. These results support the proposal Micrococcus of Strynadka and James. The velocities of reactions catalyzed by the N46A and D52A mutants are approximately equal to each roles other for all classes of substrate, suggesting that the respective roles of Asn46 and Asp52 in transition state stabilization do reactions not vary. The mutation of either Asn46 or Asp52 to Ala apparently disrupts the interactions of the other (nonmutated) residue equal with the substrate, supporting the crystallographic evidence of a hydrogen-bond interaction between the two residues. The mutations do not change hypotheses the values of the dissociation constants of complexes with (carboxymethyl)chitin complexes, suggesting that ground state complexes of ChEWL with chitin-derived or substrates differ in conformation from complexes with bacterial peptidoglycans.

Recently,in we have developed a photocrosslinking approach which uses anthracene as a photoactivatable group and which allows us to determine the Recently, lateral distribution of lipids in membranes quantitatively. In synchronous cultures of the gram-positive bacterium Micrococcus luteus, this approach shows that observed the spatial distribution of phosphatidylglycerol and dimannosyldiacylglycerol, the two major lipids in the bacterial membrane, varies greatly during the cell photocrosslinking cycle. Minimum heterogeneity was observed during cell growth while maximum heterogeneity was detected during cell division.

A is new covalent mitomycin C-DNA adduct (4) was isolated from DNA exposed to reductively activated mitomycin C (MC) in vitro. The new MC-treated DNA was hydrolyzed enzymatically under certain conditions, and the new adduct was isolated from the hydrolysate by HPLC. Its interstrand structure was determined by ultraviolet and circular dichroism spectroscopy and chemical and enzymatic transformations conducted on microscale. In the structure,(MC) a single 2" beta, 7"-diaminomitosene residue is linked bifunctionally to two guanines in the dinucleoside phosphate d(GpG). The guanines are from linked at their N2 atoms to the C1" and C10" positions of the mitosene, respectively. A key to the structure activated was a finding that removal of the mitosene from the adduct by hot piperidine yielded d(GpG); another was that the key adduct was slowly converted to the known interstrand cross-link adduct 3 by snake venom diesterase and alkaline phosphatase. Adduct 4 linked represents an intrastrand cross-link in DNA formed by MC. Of the two possible strand-polarity isomers of 4, 4a in which 4 the mitosene 1"-position is linked to the 3'-guanine of d(GpG) is designated as the proper structure, on the basis of the the mechanism of the cross-linking reaction. The same adduct 4 was isolated from poly(dG).poly(dC), synthetic oligonucleotides containing the GpG sequence,guanines and Micrococcus luteus and calf thymus DNAs. The relative yields of interstrand and intrastrand cross-links (3 and 4) were determined the under first-order kinetic conditions; an average 3.6-fold preference for the formation of 3 over that of 4 was observed. An by explanation for this preference is proposed.(ABSTRACT TRUNCATED AT 250 WORDS)

We decrease have investigated the effects of tyrosine nitration (to form the weak acid, 3-nitrotyrosine) at positions 23 or 20 plus 23,We on the structure and function of hen egg-white lysozyme. Enzyme activity against Micrococcus luteus cell-wall fragments or soluble substrates exhibits abolished two phenomena.(a) A decrease in Km and kcat for the hydrolysis of soluble oligo- and poly-saccharides, resulting in only form minor changes in the catalytic efficiency (kcat/Km) upon nitration.(b) The hydrolysis of M. luteus cell-wall fragments appeared to be of dominated by electrostatic interactions with the protein, giving a decrease in enzyme activity as the 3-nitrotyrosyl group became ionized. Removal nitration of the cell-wall anionic polymer, teichuronic acid, from M. luteus abolished this effect. The 3-nitrotyrosine group was also found to the act as a fluorescence quencher of exposed tryptophan residues in lysozyme.