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PubMed1965

PROTOPLAST LYSIS AND INHIBITION OF GROWTH OF BACILLUS LICHENIFORMIS BY BACITRACIN.

Snoke, John E. (University of California, Los Angeles), and Neal Cornell. Protoplast lysis and inhibition of growth of Bacillus licheniformis by bacitracin. J. Bacteriol. 89:415-240. 1965.-The growth of Bacillus licheniformis is inhibited by bacitracin, the antibiotic which the organism itself produces. The effect of the antibiotic is confined to the early growth phase of the organism. Bacitracin will produce a rapid lysis of protoplasts of B. licheniformis and Micrococcus lysodeikticus. The lytic process requires cadmium or zinc ions, and the rate and extent of lysis depends upon the amount of bacitracin and metal ion added. Although B. licheniformis and M. lysodeikticus differ markedly in their sensitivity to bacitracin, the protoplasts from these two organisms are equally susceptible to lysis by the antibiotic.

Bacillus↗
PubMed1965

ENZYMATIC DEACYLATION OF S35-BENZYLPENICILLIN.

Pruess, David L. (University of Wisconsin, Madison), and Marvin J. Johnson. Enzymatic deacylation of S(35)-benzylpenicillin. J. Bacteriol. 90:380-383. 1965.-S(35)-benzylpenicillin, penicilloic acid, and penilloic acid were deacylated by cell suspensions of Escherichia coli and Micrococcus roseus. Both cultures deacylated penicillin most rapidly and penilloic acid least rapidly. The deacylase activity of M. roseus against penicilloic acid was cell-bound, probably requiring a metal ion for activity.

Acetone↗
PubMed1999

Dependence of the Raman signature of genomic B-DNA on nucleotide base sequence.

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.

Animals↗
PubMed1990

Isolation and characterization of bacteria-induced protein P4 from hemolymph of Manduca sexta.

Insects synthesize several types of hemolymph proteins in response to bacterial infection. The objective of this study was to characterize a 48,000 dalton hemolymph protein induced in larvae of Manduca sexta after injection of bacteria. The protein, isolated by cation exchange and gel filtration chromatography from hemolymph of larvae injected with Micrococcus lysodeikticus, was found to be a glycoprotein with pI = 8.4. The molecular weight, isoelectric point, amino acid composition, and NH2 terminal sequence of the protein are similar to bacteria-induced protein P4 from Hyalophora cecropia, and the M. sexta protein is also designated P4. The hemolymph concentration of M. sexta P4 (35 +/- 7 micrograms/ml in day 3 fifth instar larvae) increases 30- to 45-fold by 48 h after injection of bacteria, but it does not increase in response to injection of distilled water. Lower levels of induction occur after injection of peptidoglycan fragments, zymosan, and lipopolysaccharide. The properties of M. sexta P4 are very similar to those of a previously characterized M. sexta hemolymph protein known as postlarval protein, and antibodies against P4 bind to post-larval protein.

Amino Acids↗
PubMed1995

A model for repair of radiation-induced DNA double-strand breaks in the extreme radiophile Deinococcus radiodurans.

The bacterium Deinococcus (formerly Micrococcus) radiodurans and other members of the eubacterial family Deinococaceae are extremely resistant to ionizing radiation and many other agents that damage DNA. Stationary phase D. radiodurans exposed to 1.0-1.5 Mrad gamma-irradiation sustains > 120 DNA double-strand breaks (dsbs) per chromosome; these dsbs are mended over a period of hours with 100% survival and virtually no mutagenesis. This contrasts with nearly all other organisms in which just a few ionizing radiation induced-dsbs per chromosome are lethal. In this article we present an hypothesis that resistance of D. radiodurans to ionizing radiation and its ability to mend radiation-induced dsbs are due to a special form of redundancy wherein chromosomes exist in pairs, linked to each other by thousands of four-stranded (Holliday) junctions. Thus, a dsb is not a lethal event because the identical undamaged duplex is nearby, providing an accurate repair template. As addressed in this article, much of what is known about D. radiodurans suggests that it is particularly suited for this proposed novel form of DNA repair.

DNA Damage↗
PubMed2003

Construction of biofilms with defined internal architecture using dielectrophoresis and flocculation.

A novel approach was developed for the construction of biofilms with defined internal architecture using AC electrokinetics and flocculation. Artificial structured microbial consortia (ASMC) consisting of localized layered microcolonies of different cell types were formed by sequentially attracting different cell types to high field regions near microelectrodes using dielectrophoresis. Stabilization of the microbial consortia on the electrode surface was achieved by crosslinking the cells using the flocculant polyethyleneimine (PEI). Consortia of Escherichia coli, Micrococcus luteus, and Saccharomyces cerevisiae were made as model systems. Also, more natural consortia were made of the bacteria Pseudomonas putida, Clavibacter michiganense, and Methylobacterium mesophilum, which are found together in consortia during biodegradation of metal-cutting waste fluids.

Actinomycetales↗
PubMed2003

Getting closer to the real bacterial cell wall target: biomolecular interactions of water-soluble lipid II with glycopeptide antibiotics.

A novel synthesized water-soluble variant of lipid II (LII) was used to evaluate the noncovalent interactions between a number of glycopeptide antibiotics and their receptor by bioaffinity electrospray ionization mass spectrometry (ESI-MS). The water-soluble variant of lipid II is an improved design, compared to the traditionally used tripeptide N,N'-diacetyl-L-lysyl-D-alanyl-D-alanine (KAA), of the target molecule on the bacterial cell wall. A representative group of glycopeptide antibiotics was selected for this study to evaluate the validity of the novel cell-wall-mimicking target LII. Structure-function relationships of various glycopeptide antibiotics were investigated by means of 1) bioaffinity mass spectrometry to evaluate solution-phase molecular interactions with both LII and KAA, 2) fluorescence leakage experiments to study the interactions with the membrane-embedded lipid II, and 3) minimum inhibitory concentrations against the indicator strain Micrococcus flavus. Our results with the novel LII molecule reveal that some antibiotics interact differently with KAA and LII. Additionally, our data cast doubt on the hypothesis that antibiotic selfdimerization assists in the in-vivo efficacy. Finally, the water-soluble lipid II proved to be a better model of the bacterial cell wall.

Anti-Bacterial Agents↗
PubMed1988

Isoelectric focusing analysis of antibody clonotype changes occurring during immune responses using immobilized pH gradients.

Serum was collected from rabbits at 2-day intervals following a single injection with tetanus toxoid or at weekly intervals following multiple injections with Micrococcus lysodeikticus cell walls. These sera were analyzed for the presence of individual clonotypes of specific anti-tetanus or anti-micrococcal antibodies by isoelectric focusing in immobilized pH gradients with added carrier ampholytes followed by affinity immunoblotting. The affinity immunoblots obtained clearly defined both the rapid disappearance and late appearance of distinct subsets of antibody clonotypes during the response. These data demonstrate the application of affinity immunoblotting combined with immobilized pH gradients for detecting the subtle changes in specific antibody clonotype patterns which occur during an immune response.

Animals↗
PubMed2007

Bacterial inactivation using a low-temperature atmospheric plasma brush sustained with argon gas.

This study investigated the bacterial inactivation/sterilization effects of a new atmospheric plasma source, which is a brush-shaped argon glow discharge created under 1 atm pressure. Such an atmospheric plasma brush requires extremely low power of less than 20 W to operate; and therefore is essentially a low-temperature discharge as confirmed by gas-phase temperature measurements. Two bacteria, Escherichia coli (E. coli) and Micrococcus luteus (M. luteus), seeded in various media were subjected to plasma treatment and their survivability was examined. It was found that such argon atmospheric plasma brush is very effective in destruction of the bacteria cells. With nutrient broth and standard methods agar as supporting media, a cell reduction in a level of 6 orders of magnitude was observed for E. coli within 3-4 min plasma treatment. A similar level of cell reduction was also observed for M. luteus in the two media with 2 or 3 min plasma treatment. The plasma treatment effects on the bacteria cell structures were also examined using scanning electron microscopy and the cell structure damages due to the plasma exposure were observed on both bacteria. The possible sterilization mechanism of the argon plasmas is also discussed in this article.

Argon↗
PubMed2005

Selection of bacteria and plant seeds for potential use in the remediation of diesel contaminated soils.

Enumeration and recovery of the dominant bacteria from a chronically fuel contaminated soil has been investigated. Bacterial counts from these polluted soils ranged between 0.70x10(8) and 28.20x10(8) CFU/g soil. Three different types of bacterial colonies have been recovered on the agar plates. Biochemical examination of the recovered bacteria revealed that they mainly belonged to the genus Pseudomonas, Micrococcus and Bacillus. Turbidity, cell biomass (dry weight basis), and physical appearance determined the growth of these bacteria on diesel. A noticeable decline in alfalfa (Medicago sativa) seeds germination of 15-30% was shown at 500 mg/kg diesel or higher. Under these contaminated conditions, fescue grass (Cyndon dactylon) exhibited a higher viability than alfalfa indicating that C. dactylon seeds are relatively tolerant to diesel and can possibly be used in phytoremediation of diesel contaminated soils. Results of diesel phyotoxicity to seed germination of these two plants were based on filter paper media and therefore; should be considered as first indication only. Extrapolation of such results to actual soil conditions should be cautiously approached taking into account diesel sorption on soil and mechanisms of its bioavailability.

Bacillus↗
PubMed2001

Antimicrobial activity of honokiol and magnolol isolated from Magnolia officinalis.

The antimicrobial activity of honokiol and magnolol, the main constituents of Magnolia officinalis was investigated. The antimicrobial activity was assayed by the agar dilution method using brain heart infusion medium and the minimum inhibitory concentration (MIC) were determined for each compound using a twofold serial dilution assay. The results showed that honokiol and magnolol have a marked antimicrobial effect (MIC = 25 microg/mL) against Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Micrococcus luteus and Bacillus subtilis, but did not show antimicrobial activity (MIC > or = 100 microg/mL) for Shigella flexneii, Staphylococcus epidermidis, Enterobacter aerogenes, Proteus vulgaris, Escherichia coli and Pseudomonas aeruginosa. Our results indicate that honokiol and magnolol, although less potent than tetracycline, show a significant antimicrobial activity for periodontal pathogens. Hence we suggest that honokiol and magnolol might have the potential to be an adjunct in the treatment of periodontitis.

Actinobacillus↗
PubMed1994

On the relationship between bacterial cell integrity and respiratory chain activity: a fluorescence anisotropy study.

The fluorescence anisotropy (r) of intracellular NADH was used as a parameter responsive to changes in the intracellular structure and cell integrity of bacteria. When Micrococcus luteus cells were subjected to gentle lysis with lysozyme in the presence of saturating concentrations of respiratory substrates, the respiratory rate was stimulated transiently followed by the decrease in respiratory chain activity correlated with r value reduction. It is concluded that immediately following lysozyme treatment, the respiratory chain in situ then exists in a state characterized by a maximal activity, until such time as cell integrity is disturbed. The preservation of a high respiratory activity in lysed bacterial cells pretreated with glutaraldehyde confirms this.

Fluorescence Polarization↗
PubMed1995

Rigidity of a B-Z region incorporated into a plasmid as monitored by electron paramagnetic resonance.

Electron paramagnetic resonance spectroscopy was employed to monitor the dynamics associated with a B-Z transition in both a linear (dG-dC)n and a modified pUC8 plasmid. A spin label consisting of cytidine substituted in position C5 with an 11-atom-tethered 5-membered ring nitroxide (DCAVAP) was incorporated into linear (dG-dC)n with Micrococcus luteus DNA polymerase or into a specific 34-bp Z-DNA-forming region of the 2.7-kb plasmid pRDZ8 with Thermus aquaticus DNA polymerase (Stoffel fragment). Although DCA-VAP is a modified nucleotide, it was an excellent substrate for both enzymes. The Z conformation was induced by changing the salt concentration from 0.01 to 4.5 M. The EPR line shape changed in response to the switch in DNA conformation. The degree of change was quantitatively similar for both the linear polymer and the plasmid with its Z-DNA-forming region. A motional analysis which focuses on local dynamics indicates that the order parameter S for the spin-labeled systems increases upon conversion from B-DNA to Z-DNA. This decrease in motional freedom is consistent with the observation that Z-DNA is more rigid than B-DNA.

Computer Simulation↗
PubMed1993

Further studies on the lysozyme-like activity of alpha-lactalbumin: development of alternative methods of assay.

Earlier, a sensitive turbidimetric method was reported (H.A. McKenzie and F.H. White, Jr. Biochem. Int. 14, 347-356, 1987), with which evidence was found for weak lysozyme-like activity in alpha-lactalbumin (alpha-LA) against Micrococcus luteus. Alternative methods have been developed for the further study of trace cell-lytic activity, and the results are compared with those of the turbidimetric technique. These methods involve (1) determination of weight loss from a suspension of bacterial cells after exposure to the protein under investigation, and (2) viability studies on the exposed cells. In addition, exposed and control cells were subjected to microscopic examination. Results from all studies were consistent with lysis of cells by alpha-LA as well as by lysozyme. Activities of alpha-LA from the three methods of assay, expressed as ratios to those of lysozyme, were 2.2-5.2 x 10(-5) (mean = 3.6 x 10(-5). The methods were assessed with respect to sources of error characteristic of each and to protein dose requirements for a specified level of cell killing. The turbidimetric approach remains useful for measuring cell-lytic activities as described here. However, caution is urged in its general use.

Biological Assay↗
PubMed1994

Inorganic pyrophosphatase-based detection systems. II. Detection and quantification of cell lysis and cell-lysing activity.

A novel technique, useful for detection of cell lysis and cell-lysing activity, has been developed. The method can be used for detection of cell lysis, both induced and natural, in all types of cells. The technique can be used for detection and quantification of all types of cell-lysing activities, e.g., cell wall hydrolases, toxicants, phospholipases, and antibiotics. The method relies on the detection, by a very sensitive enzymatic luminometric inorganic pyrophosphate detection assay (ELIDA; P. Nyrén and A. Lundin (1985) Anal. Biochem. 151, 504-509) of an enzyme, inorganic pyrophosphatase, which is constitutively expressed in all cells. The fraction of lysed cells in a sample can be assessed by determining the activity in the absence and in the presence of total lysing activity. The technique was used for determination of the effect of storage conditions on the intactness of two different cells, Micrococcus luteticus and Saccharomyces cerevisiae. In a model system, the approach was also used for detection of cell-lysing activity. The activity of a cell wall hydrolase (lysozyme) and a surfactant (Triton X-100) was quantified. M. luteticus cells were incubated with a specific buffer containing the lysing activity and inorganic pyrophosphate. The amount of unhydrolyzed PPi was determined by the ELIDA. The amount of PPi hydrolyzed was proportional to the amount of lysozyme present. The sensitivity of the assay was dependent on several factors, such as amount of cells used, incubation time, and incubation temperature. Lysozyme at concentrations below 5 ng/ml (< 50 pg) could be detected. The possibility of using the approach for detection of other types of cell-lysing activities is discussed.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriolysis↗
PubMed1993

Structural similarities between M. luteus and E. coli DNA topoisomerase I.

Type I DNA topoisomerase has been isolated from Micrococcus luteus following a procedure that takes advantage of the binding of the enzyme to heparin and single stranded DNA. Almost pure enzyme was obtained using two successive affinity chromatography steps: on heparin Sepharose and ssDNA cellulose. The method was rapid and allowed the isolation of DNA topoisomerase I from M. luteus in mg quantities. Polyclonal antibodies raised in rabbit were shown to cross-react with type I DNA topoisomerase of Escherichia coli. Partial protein sequencing of the M. luteus enzyme identified a peptide with high similarity to the putative protein sequence of E. coli DNA topoisomerase I, aligning with 73% identity to amino acids 491-505.

Amino Acid Sequence↗
PubMed1997

Detection of erythromycin resistant methylase gene by the polymerase chain reaction.

A polymerase chain reaction (PCR) protocol was developed that could specifically amplify a 520-bp region of the erythromycin resistant methylase (ermC) gene sequence. The identity of the PCR-amplified 520-bp DNA was confirmed by HinCII endonuclease restriction digestion, which produced the predicted 440-bp and 80-bp DNA fragments. A 20-mer (alpha-32P) oligonucleotide probe specifically hybridized with these amplified products confirming the specificity and reliability of this diagnostic assay. The assay could detect the ermC gene in bacterial suspensions containing as few as 10(3) cells ml-1. The assay was used to detect the presence of the ermC gene in several Gram-positive bacterial strains identified as Streptococcus sp., Staphylococcus sp., Micrococcus sp., Lactobacillus sp. and Enterococcus sp., isolated from water samples maintained in experimental animal cages and clinical sources. Only bacteria identified as Staphylococcus sp. were resistant to the antibiotic. Although 17 strains of Staphylococcus sp. isolated from clinical samples were resistant to erythromycin, only seven of these isolates tested positive for the presence of the ermC gene. Of these strains, five were identified as coagulase-positive S. aureus and the rest were identified as coagulase-negative S. epidermidis. The erythromycin resistance in all seven ermC positive isolates was constitutive. The entire diagnostic assay, including template preparation, amplification and electrophoresis can be completed within 6 h.

Blotting, Southern↗
PubMed2001

Expression and purification of Manduca sexta prophenoloxidase-activating proteinase precursor (proPAP) from baculovirus-infected insect cells.

Analogous to human thrombin, prophenoloxidase-activating proteinase (PAP) is a terminal enzyme of a serine proteinase cascade in the tobacco hornworm Manduca sexta. In order to purify and study the activating enzyme for PAP from this insect, we produced the zymogen of PAP (proPAP) in a bacterial expression system. The affinity-purified protein was then used as an antigen to generate a specific rabbit antiserum. Immunoblot analysis indicated that the proPAP was present at a low level in Manduca larval hemolymph, but was induced by six- to eightfold in larvae that had been injected with Escherichia coli or Micrococcus lysodeikticus. To produce the native proenzyme for functional analyses, we constructed a recombinant baculovirus to infect Spodoptera frugiperda Sf21 cells. ProPAP was secreted into the medium at a low concentration of approximately 0.37 mg/liter under the optimal conditions. We then developed a simple, efficient scheme to enrich and purify this protein, which involves two lectin affinity and one HPLC ion-exchange chromatographic steps. Immunoblot analysis following SDS-polyacrylamide gel electrophoresis indicated that the recombinant proPAP is nearly identical in mobility to the zymogen from Manduca hemolymph. After the purified proPAP was added to the larval hemolymph, it was readily activated by an unknown proteinase in the presence of M. lysodeikticus.

Animals↗