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

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

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

Elongation factors G, Tu, and related proteins (including LepA) form a distinct subgroup within the GTPase superfamily. This observation is based primarily upon amino acid comparisons of the effector region (G2) of the GTP-binding domain. To examine the functional importance of the highly conserved elongation factor G2 domain a series of chimeric proteins were constructed between Escherichia coli EF-G and Micrococcus luteus EF-G, and between E. coli EF-G and LepA (a protein of unknown function). The M. luteus EF-G/E. coli EF-G hybrid, M. luteus EF-G, and E. coli EF-G efficiently complemented EF-G function in an E. coli strain (PEM101) harbouring a temperature-sensitive mutation in fusA (the gene encoding EF-G). A comparison of the amino acid sequences of the M. luteus EF-G and E. coli EF-G indicated that groups of divergent amino acid residues (amino acids 1-9 and 72-80) were not important for function. LepA and LepA/EF-G chimeric proteins were tested for the ability to complement EF-G function in vivo, for cross-linking to 8-azido-[gamma-32P]-GTP in vitro and for fusidic acid-dependent co-sedimentation with 70S ribosomes. With one exception, all chimeras could be readily cross-linked to azido-GTP in an EF-G-like manner, indicating that hybrid protein construction did not generally result in improperly folded GTP-binding domains. However, the inability of such chimeras to complement EF-G function in vivo indicates that the effector domains are not functionally interchangeable. All LepA/EF-G chimeric proteins were severely defective in fusidic acid-dependent complex formation with 70S ribosomes. A comparison of the amino acid sequences of all three proteins suggests that residues 30-33, 43-48, and 63-66 of E. coli EF-G are important for EF-G specific ribosome-associated function.

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

The vibrational spectra of four genomic and two synthetic DNAs, encompassing a wide range in base composition [poly(dA-dT). poly(dA-dT), 0% G + C; Clostridium perfringens DNA, 27% G + C; calf thymus DNA, 42% G + C; Escherichia coli DNA, 50% G + C; Micrococcus luteus DNA, 72% G + C; poly(dG-dC).poly(dG-dC), 100% G + C](dA: deoxyadenosine; dG: deoxyguanosine; dC: deoxycytidine; dT: thymidine), have been analyzed using Raman difference methods of high sensitivity. The results show that the Raman signature of B DNA depends in detail upon both genomic base composition and sequence. Raman bands assigned to vibrational modes of the deoxyribose-phosphate backbone are among the most sensitive to base sequence, indicating that within the B family of conformations major differences occur in the backbone geometry of AT- and GC-rich domains. Raman bands assigned to in-plane vibrations of the purine and pyrimidine bases-particularly of A and T-exhibit large deviations from the patterns expected for random base distributions, establishing that Raman hypochromic effects in genomic DNA are also highly sequence dependent. The present study provides a basis for future use of Raman spectroscopy to analyze sequence-specific DNA-ligand interactions. The demonstration of sequence dependency in the Raman spectrum of genomic B DNA also implies the capability to distinguish genomic DNAs by means of their characteristic Raman signatures.

Within the frame of our study we investigated Microccocus luteus, Neisseria cinerea, and Pseudomonas fluorescens by means of capillary zone electrophoresis (CZE). They form chains and clusters on a different scale, which can be reflected in the electropherograms. A low buffer concentration of Tris-borate and Na2EDTA containing a polymeric matrix of 0.0125% poly(ethylene) oxide (PEO) was used. Key factors were the standardization and optimization of CE conditions, buffer solution, and pretreatment of bacterial samples, which are not transferable to different bacterial strains, in general. The different compositions of the cell wall of on the one hand Gram-positive (M. luteus) and Gram-negative (N. cinerea) cocci and on the other hand Gram-negative, rod-shaped bacteria (P. fluorescens), are probably responsible for the different pretreatment conditions.

The biosynthesis of three mannolipids and the presence of a membrane-associated lipomannan in Micrococcus luteus (formerly Micrococcus lysodeikticus) were documented over 30 years ago. Structural and topological studies have been conducted to learn more about the possible role of the mannolipids 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 (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 acylated with a 12-methyltetradecanoyl group and the sn-2 position is acylated with a myristoyl group. The lipomannan is shown to be located on the exterior face of the cytoplasmic membrane, and not exposed on the surface of intact cells, by staining of intact protoplasts with fluorescein isothiocyanate (FITC)-linked concanavalin A (Con A). When cell homogenates of M. luteus are incubated 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 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 and consequently the lipomannan. These results strongly indicate that GDP-Man is the direct mannosyl donor for the synthesis of Man1-2-DAG, and that the majority of the 50 mannosyl units in the lipomannan are derived from Man-P-Undec. Protease-sensitivity studies with intact and lysed protoplasts indicate that the active sites of the mannosyltransferases catalyzing the formation of Man1-2-DAG and Man-P-Undec are exposed on the inner face, and the Man-P-Undec-mediated reactions occur on the outer surface of the cytoplasmic membrane. Based on all of these results, a topological model is proposed for the lipid-mediated assembly of the membrane-bound lipomannan.

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

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