Nutrition and growth of the moderately halophilic bacteria Micrococcus morrhuae K-17 and Micrococcus luteus K-15

Chemically defined media have been developed for the growth of two moderately halophilic bacteria, Micrococcus morrhuae K-17 and Micrococcus luteus K-15. M. morrhuae K-17 grows well in a synthetic medium (SM-1) which contains a number of salts, 0.21 M KCl, 2 M NaCl, D-mannose, five vitamins and ten amino acids. The synthetic medium (SM-2) for M. luteus K-15 contains a number of salts, 0.21 M KCl, 1 M NaCl, D-fructose, nine vitamins and nine amino acids. Nutritional studies show that M. morrhuae K-17 can utilize a large number of organic compounds as carbon and energy source while the ability of M. luteus K-15 in utilizing the organic compounds is rather limited. The minimum salt requirement is 0.5 M NaCl for both strains when growth at the optimum temperature of 30 degrees C. However, this requirement can be lowered to 0.2 M in M. luteus K-15 when grown at a lower temperature of 25 degrees C. It is concluded that the ability to grow in a wider range of salt concentrations in response to temperature is species specific in moderate halophiles. The salt range for growth to occur can be extended when cells of both strains are grown in complex medium which might provide the amino acids and growth factors that cannot be synthesized by these strains at high salt concentrations.     

Effect of physiological age on radiation resistance of some bacteria that are highly radiation resistant.

Physiological age-dependent variation in radiation resistance was studied for three bacteria that are highly radiation resistant: Micrococcus radiodurans, Micrococcus sp. isolate C-3, and Moraxella sp. isolate 4. Stationary-phase cultures of M. radiodurans and isolate C-3 were much more resistant to gamma radiation than were log-phase cultures. This pattern of relative resistance was reversed for isolate 4. Resistance of isolate 4 to UV light was also greater during log phase, although heat resistance and NaCl tolerance after heat stress were greater during stationary phase. Radiation-induced injury of isolate 4 compared with injury of Escherichia coli B suggested that the injury process, as well as the lethal process, was affected by growth phase. The hypothesis that growth rate affects radiation resistance was tested, and results were interpreted in light of the probable confounding effect of methods used to alter growth rates of bacteria. These results indicate that dose-response experiments should be designed to measure survival during the most resistant growth phase of the organism under study. This timing is particularly important when extrapolations of survival results might be made to potential irradiation processes for foods.     

Effect of physiological age on radiation resistance of some bacteria that are highly radiation resistant

Physiological age-dependent variation in radiation resistance was studied for three bacteria that are highly radiation resistant: Micrococcus radiodurans, Micrococcus sp. isolate C-3, and Moraxella sp. isolate 4. Stationary-phase cultures of M. radiodurans and isolate C-3 were much more resistant to gamma radiation than were log-phase cultures. This pattern of relative resistance was reversed for isolate 4. Resistance of isolate 4 to UV light was also greater during log phase, although heat resistance and NaCl tolerance after heat stress were greater during stationary phase. Radiation-induced injury of isolate 4 compared with injury of Escherichia coli B suggested that the injury process, as well as the lethal process, was affected by growth phase. The hypothesis that growth rate affects radiation resistance was tested, and results were interpreted in light of the probable confounding effect of methods used to alter growth rates of bacteria. These results indicate that dose-response experiments should be designed to measure survival during the most resistant growth phase of the organism under study. This timing is particularly important when extrapolations of survival results might be made to potential irradiation processes for foods.     

Influence of growth environment on coaggregation between freshwater biofilm bacteria

AIM: To characterize the expression of coaggregation between Blastomonas natatoria 2.1 and Micrococcus luteus 2.13 following growth in liquid culture, on agar and in an artificial biofilm matrix composed of poloxamer hydrogel. METHODS AND RESULTS: The ability of B. natatoria 2.1 and M. luteus 2.13 to coaggregate with one another was assessed following growth in liquid culture as colonies on agar or within a poloxamer hydrogel matrix. In all these environments a cycle of gain and loss of coaggregation occurred when the two cell types were aged simultaneously, with optimum expression occurring in early stationary phase. Blastomonas natatoria 2.1 cells only coaggregated maximally after entry into stationary phase. Conversely, M. luteus 2.13 cells only coaggregated in exponential phase and early stationary phase and coaggregation ability was lost in late stationary phase. Maximal coaggregation therefore only occurred between the two strains if both were in early stationary phase, when the surface properties of the two cell types were optimal for coaggregation. CONCLUSION: In addition to occurring between cells grown in liquid culture, coaggregation between aquatic bacteria occurs after growth as a biofilm on agar and in an artificial biofilm matrix in poloxamer. Under all conditions, the B. natatoria 2.1 coaggregation adhesin and complementary receptor on M. luteus 2.13 were only expressed simultaneously during early stationary phase.     

Amino acid sequence of the murein of Planococcus and other Micrococcaceae

The amino acid composition and amino acid sequence of the murein (peptidoglycan) of 10 strains of planococci were studied. It is shown that the peptide subunit consists of muramyl-l-alanyl-gamma-d-glutamyl-l-lysyl-d -alanine. The cross-linking of two adjacent peptide subunits is mediated by d-glutamic acid which is bound to the epsilon-amino group of lysine by its gamma-carboxyl group and to the carboxyl group of d-alanine of an adjacent peptide subunit by its amino group. About 20 to 25% of the peptide subunits are not cross-linked. The murein structure of the different species and strains of Micrococcus, Staphylococcus, and Sarcina are compared. It is evident that the murein structure is a very good criterion for grouping the micrococci. In addition, some of these groups are fairly well defined by physiological properties as well as by their guanine + cytosine content of the deoxyribonucleic acid e.g., Micrococcus, Staphylococcus, Planococcus, Sarcina ureae. Other groups, represented by a single or a few strains only, such as M. varians NTCC 7281, M. radiodurans, M. freudenreichii ATCC 407, and M. luteus ATCC 398, need further investigation.     

Occurrence of bacteria in the mouth from genera of Micrococcus,Kocuria, Nesterenkonia,Kytococcus and Dermacoccus

The aim of the study was to assess the prevalence of different bacteria in the oral cavity. The bacteria were present in the oral cavities of 73 (48.7%) of 150 individuals. Nesterenkonia halobia, the most frequently isolated species, was found in 20 (27%) individuals, Micrococcus luteus in 16 (22%), Kocuria kristinae in 12 (16%), Kocuria varians in 10 (14%), Dermacoccus sedentarius in 9 (12%), Micrococcus lylae in 8 (11%), and Kytococcus nishinomiyaensis in 3 (4%). Mean counts of these microorganisms were relatively low and amounted in log10 CFU/ml saliva for M. luteus 1.87 +/- 0.52, for M. lylae 2.03 +/- 0.39, for N. halobia 2.14 +/- 0.56, for K. kristinae 2.20 +/- 0.69, for K. varians 2.19 +/- 0.67, for K. nishinomiyaensis 1.72 +/- 0.39, and for D. sedentarius 2.27 +/- 0.55. The factor limiting the population sizes of these microorganisms was most probably the antagonistic activity of the bacteria living in oral cavity.     

Deinococcus radiodurans UV endonuclease beta DNA incisions do not generate photoreversible thymine residues

The ability of UV endonuclease beta of Deinococcus radiodurans to act as a pyrimidine dimer DNA glycosylase was investigated. Cell-free extracts of D. radiodurans exhibiting UV endonuclease beta activity failed to generate incisions in irradiated DNA that liberated free-thymine residues upon photoreversal with 254-nm light. This is in marked contrast to the pyrimidine dimer UV glycosylase of Micrococcus luteus that does liberate such residues. The result suggests that UV endonuclease beta incises DNA by true endonuclease action.     

Metabolism of pyrimidine bases and nucleosides in the coryneform bacteria Brevibacterium ammoniagenes and Micrococcus luteus.

The metabolism of exogenous pyrimidine bases and nucleosides was investigated in Brevibacterium ammoniagenes and Micrococcus luteus with fluorinated analogs and radioactive precursors. Salvage of thymine and thymidine was found in M. luteus, but not in B. ammoniagenes. Exogenous uracil or uracil nucleosides, but not cytosine or cytosine nucleosides, were nucleic acid precursors for both bacteria. By examining the possible nucleoside-metabolizing enzymes, it can be suggested that the pyrimidine salvage pathways in the coryneform bacteria are different from those of members of the family Enterobacteriaceae.     

Growth, encystment and survival of Acanthamoeba castellanii grazing on different bacteria

Free-living amoebae of the genus Acanthamoeba are widely distributed in soil and water collections, where trophozoites (vegetative, multiplicative stages) feed mainly by phagocytosis and thus control bacterial populations in the environment. Here, we examined the growth, encystment and survival of Acanthamoeba castellanii receiving different bacteria (Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, Bacillus subtilis, Bacillus megaterium, Micrococcus luteus, and Staphylococcus aureus) in nonnutrient saline. All bacteria assayed induced a dose-dependent proliferative response, in most cases maximized with a bacterial dose of 1 x 10(9) mL(-1); except for M. luteus, trophozoites grew better with viable than with heat-killed bacteria. In addition, Acanthamoeba growth was improved by adding bacteria on alternate days. Single-dose experiments indicated a temporal association between the growth of trophozoite and bacterial consumption, and higher consumption of M. luteus, E. coli and P. aeruginosa, bacterial species that allowed the highest trophozoite yields. Long-term Acanthamoeba-bacteria incubation revealed that encystment was significantly delayed by almost all the bacteria assayed (including S. aureus, which elicited a poor growth response) and that the presence of bacteria markedly increased cyst yield; final cyst recovery clearly depended on both the dose and the type of the bacterium given, being much higher with E. coli, M. luteus and P. aeruginosa.     

Repair of ultraviolet light-induced damage in Micrococcus radiophilus, an extremely resistant microorganism.

Repair of ultraviolet radiation damage was examined in an extremely radioresistant organism, Micrococcus radiophilus. Measurement of the number of thymine-containing dimers formed as a function of ultraviolet dose suggests that the ability of this organism to withstand high doses of ultraviolet radiation (20,000 ergs/mm2) is not related to protective screening by pigments. M. radiophilus carries out a rapid excision of thymine dimers at doses of ultraviolet light up to 10,000 ergs/mm2. Synthesis of deoxyribonucleic acid is reduced after irradiation, but after removal of photodamage the rate approaches that in unirradiated cells. A comparison is drawn with Micrococcus luteus and M. radiodurans. We conclude that the extremely high resistance to ultraviolet irradiation in M. radiophilus is at least partly due to the presence of an efficient excision repair system.     

The role of alkylhydroxybenzenes in the adaptation of Micrococcus luteus to heat shock

The response of the gram-positive bacterium Micrococcus luteus to heat shock (45 degrees C, 15 min) and the adaptogenic activity of alkylhydroxybenzenes (AHB), which are extracellular growth-regulating substances of these bacteria, were studied. The perception of stress and the postshock behavior of M. luteus cells proved to depend on the growth phase and medium. The magnitude of stress response was more pronounced in cultures grown on synthetic medium than in cultures grown on rich medium (nutrient broth). During exponential or linear growth, the cells were more sensitive to the temperature effect than during decelerated growth. In linearly growing M. luteus cultures, the amount of total intra- and extracellular alkylhydroxybenzenes, the anabiosis inducers, increased in response to heat shock. AHB redistribution between cells and culture liquid occurred in the course of stress and after stress. In micrococci exposed to heat shock, an increase in the AHB concentration both in cells and culture liquid is likely a defense reaction of stress resistance. This conclusion was confirmed in the experiments with the addition 30 min before the heat shock of a chemical analogue of the anabiosis inducer, C7-AHB (12 mM), which protected M. luteus cells so that their intense growth was observed after shock without any lag. The protective effect of AHB is a result of their ability to form complexes with enzyme macromolecules and stabilize them. The data obtained extend the knowledge of the stress-protective functions of low-molecular-weight autoregulators and of the role of intercellular communications in the stress response of bacterial cultures.     

Degradation of anthracene by bacteria isolated from oil polluted tropical soils

Four bacteria, identified as Pseudomonas aeruginosa, Alcaligenes eutrophus, Bacillus subtilis and Micrococcus luteus were isolated from crude oil polluted soils using anthracene as the sole carbon and energy source. All the organisms utilized n-hexadecane, n-tetradecane, diesel oil, engine oil and naphthalene as sole carbon sources. None could utilize hexane, cycloheptane, xylene, benzene, toluene, phenol, fluoranthene,and kerosene as carbon sources. Highest cell density obtained with 0.1% (w/v) anthracene were 4.5 x 10(7) (cfu/ml), 8.6 x 10(6) (cfu/ml), 5.4 x 10(6) and 2.4 x 10(6) (cfu/ml) respectively, for P. aeruginosa, A. eutrophus, B. subtilis and M. luteus after 30 days incubation. Growth of the organisms on a Nigerian crude oil resulted in a residual oil concentration of 22.2%, 33.3%, 39.3%, 44% and 91.7% respectively, for P. aeruginosa, A. eutrophus, B. subtilis, M. luteus and the noninoculated control on the 14 th day. Ring fission enzymes of the meta pathway were detected in induced cells of P. aeruginosa and A. eutrophus while ortho pathway enzymes were detected in B. subtilis and M. luteus. P. aeruginosa and A. eutrophus had specific catechol-2,3-dioxygenase activities of 3.8 +/- 0.183 and 0.64 +/- 0.032 micromol/min x mg protein respectively while catechol-1,2-dioxygenase activities of 1.95 +/- 0.029 and 1.89 +/- 0.026 micromol/min x mg protein were detected in B. subtilis and M. luteus respectively. This work, highlights the capability of these unreported tropical strains of A. eutrophus, B. subtilis and M. luteus as anthracene degraders.     

Structure of the dnaA region of Micrococcus luteus: conservation and variations among eubacteria

A phylogenetic tree constructed by 5S rRNA analysis is composed of three major branches in eubacteria: high G + C Gram+, low G + C Gram+ and Gram- [Hori and Osawa, Mol. Biol. Evol. 4 (1987) 445-472]. We have shown that the characteristic dnaA region is common among Escherichia coli (Gram-), Pseudomonas putida (Gram-), and Bacillus subtilis (low G + C Gram+). We have now determined the structure of the dnaA region of Micrococcus luteus, as a representative of the last branch, high G + C Gram+. The dnaA gene and at least three other genes, rnpA, rpmH and dnaN were found to be conserved in M. luteus. Large nontranslatable regions were found flanking the dnaA gene. The upstream region is conserved in the four bacteria so far examined. On the other hand, the downstream region is conserved only in Gram+ bacteria, M. luteus and B. subtilis. The consensus sequence of the DnaA box in M. luteus seems to be TTGTCCACA, in contrast to TTATCCACA of other bacteria. These results confirm our hypothesis that the dnaA region is the replication origin of the ancestral bacteria and that the essential feature of the DnaA protein and DnaA-box combination is conserved in eubacteria.     

A family of autocrine growth factors in Mycobacterium tuberculosis

Mycobacterium tuberculosis and its close relative, Mycobacterium bovis (BCG) contain five genes whose predicted products resemble Rpf from Micrococcus luteus. Rpf is a secreted growth factor, active at picomolar concentrations, which is required for the growth of vegetative cells in minimal media at very low inoculum densities, as well as the resuscitation of dormant cells. We show here that the five cognate proteins from M. tuberculosis have very similar characteristics and properties to those of Rpf. They too stimulate bacterial growth at picomolar (and in some cases, subpicomolar) concentrations. Several lines of evidence indicate that they exert their activity from an extra-cytoplasmic location, suggesting that they are also involved in intercellular signalling. The five M. tuberculosis proteins show cross-species activity against M. luteus, Mycobacterium smegmatis and M. bovis (BCG). Actively growing cells of M. bovis (BCG) do not respond to these proteins, whereas bacteria exposed to a prolonged stationary phase do. Affinity-purified antibodies inhibit bacterial growth in vitro, suggesting that sequestration of these proteins at the cell surface might provide a means to limit or even prevent bacterial multiplication in vivo. The Rpf family of bacterial growth factors may therefore provide novel opportunities for preventing and controlling mycobacterial infections.     

Antibacterial activity in the hemolymph of myriapods (Arthropoda)

The hemolymphs of two diplopod (Chicobolus sp. and Rhapidostreptus virgator) and two chilopod species (Lithobius forficatus and Scolopendra cingulata) were tested for the presence of antibacterial substances using Petri dish tests. The native hemolymph of all species had substances acting on living Micrococcus luteus, whereas only Rhapidostreptus, Scolopendra, and Lithobius were effective against lyophilized Micrococcus. The antibacterial activity against living Micrococcus increased after inoculation with bacteria (Enterobacter cloacae beta-12) in Chicobolus and Rhapidostreptus and also against lyophilized Micrococcus in the latter. Thus, these effects appear to be inducible. None of the myriapods tested had any bacteriostatic effect on Escherichia coli D-31 whereas the growth of gram-negative E. cloacae was inhibited. The antibacterial substances in the diplopod species were unstable when heated but were resistant to freezing. At least two antibacterial substances (a lysozyme-like one and another substance) are considered to occur in Myriapoda.     

Involvement of Escherichia coli K-12 recombination functions in elimination of multicopy plasmid R6K by DNA-damaging agents

Abstract A neutral lipomannan has been isolated from the membranes of Micrococcus agilis . In contrast to the lipomannans from 2 strains of Micrococcus luteus , which contained succinic acid ranging from 5.1%–8.0%, the M. agilis lipomannan had no detectable succinyl residues and exhibited neutral behaviour on Concanavalin A-agarose rocket electrophoresis. As with the M. luteus lipomannans, mannose was the only sugar detectable (as alditol acetate) by GLC analysis in the purified M. agilis lipomannan. Fatty acids accounted for 2% of the M. agilis lipomannan and were predominantly C₁₅ branched-chain acids, with higher amounts of C₁₆ iso and C₁₇ anteiso than that found in the M. luteus polymers. Neither conditions of growth of the organism nor the method of membrane preparation appeared to be responsible for the absence of succinyl residues. This appears to be the first report of a neutral membrane amphiphile.     

The sandfly Lutzomyia longipalpis shows specific humoral responses to bacterial challenge

Abstract The presence of immune molecules induced by microorganisms in the haemolymph of Lutzomyia longipalpis sandflies has been investigated. Injections of Escherichia coli and Micrococcus luteus into female sandflies induced anti-bacterial activity in the haemolymph. Inhibition zone assays showed that haemolymph from E.coli and M.luteus injected sandflies differentially inhibited M.luteus growth. This differential effect was specific to M. luteus infection since anti-Zs. coli activity was similar in haemolymph from both E.coli oxM.luteus injected sandflies. Haemolymph following injection of either bacteria showed the induction of a 4kDa peptide. Haemolymph from M.luteus injected sandflies also contained a 33 kDa polypeptide which was absent in haemolymph from E.coli and control uninfected insects. Sandflies, in common with other insects, were shown to possess general and specific humoral immune responses to the presence of microorganisms.     

Effects of nitric oxide and nitrogen dioxide on bacterial growth

The effects of low concentrations of nitric oxide (NO) and nitrogen dioxide (NO2) on actively dividing cultures of Staphylococcus aureus, Micrococcus luteus, Micrococcus roseus, Serratia marcescens, Bacillus subtilis, Bacillus circulans, Bacillus megaterium, and Bacillus cereus were studied. Fresh cultures of each organism were incubated for 24 h at 25 degrees C on both nutrient agar and mineral salts glucose agar plates under atmospheres containing various low concentrations of NO in air (0 to 1.9 ppm [0 to 2.0 micrograms/g of air]), NO2 in air (0 to 5.5 ppm [0 to 8.8 micrograms/g of air]), or NO and NO2 in air. Bacteria grown under air only were used as controls. After incubation, the colonies that developed on the plates were counted. None of the bacteria tested was affected by NO or NO2 at the indicated concentrations while growing on nutrient agar. Serratia marcescens, B. circulans, B. subtilis, B. megaterium, and B. cereus grown on mineral salts glucose agar were not significantly affected by NO or NO2. Low concentrations (0 to 1.9 ppm) of NO were bacteriostatic to log-phase cultures of M. roseus, M. luteus, and Staphylococcus aureus grown on mineral salts glucose agar. Bacteriostatic activity over a 24-h interval was maximal at an initial NO concentration of 1 ppm. Appreciable amounts of NO2 were produced in 24 h at initial NO concentrations greater than 1 ppm. These results suggest that NO2 may reduce the bacteriostatic activity of NO. Low concentrations (0 to 5.5 ppm) of NO2 in air did not affect any of the bacteria tested. At these low concentrations, NO affected bacterial growth, although NO2, NO2-, and NO3- did not. In addition, it was determined that the bacteriostatic activity observed in this study was not due to an increase in the acidity of the medium.     

Effects of nitric oxide and nitrogen dioxide on bacterial growth.

The effects of low concentrations of nitric oxide (NO) and nitrogen dioxide (NO2) on actively dividing cultures of Staphylococcus aureus, Micrococcus luteus, Micrococcus roseus, Serratia marcescens, Bacillus subtilis, Bacillus circulans, Bacillus megaterium, and Bacillus cereus were studied. Fresh cultures of each organism were incubated for 24 h at 25 degrees C on both nutrient agar and mineral salts glucose agar plates under atmospheres containing various low concentrations of NO in air (0 to 1.9 ppm [0 to 2.0 micrograms/g of air]), NO2 in air (0 to 5.5 ppm [0 to 8.8 micrograms/g of air]), or NO and NO2 in air. Bacteria grown under air only were used as controls. After incubation, the colonies that developed on the plates were counted. None of the bacteria tested was affected by NO or NO2 at the indicated concentrations while growing on nutrient agar. Serratia marcescens, B. circulans, B. subtilis, B. megaterium, and B. cereus grown on mineral salts glucose agar were not significantly affected by NO or NO2. Low concentrations (0 to 1.9 ppm) of NO were bacteriostatic to log-phase cultures of M. roseus, M. luteus, and Staphylococcus aureus grown on mineral salts glucose agar. Bacteriostatic activity over a 24-h interval was maximal at an initial NO concentration of 1 ppm. Appreciable amounts of NO2 were produced in 24 h at initial NO concentrations greater than 1 ppm. These results suggest that NO2 may reduce the bacteriostatic activity of NO. Low concentrations (0 to 5.5 ppm) of NO2 in air did not affect any of the bacteria tested. At these low concentrations, NO affected bacterial growth, although NO2, NO2-, and NO3- did not. In addition, it was determined that the bacteriostatic activity observed in this study was not due to an increase in the acidity of the medium.     

Fourier transform IR spectroscopic appraisal of radiation damage in Micrococcus luteus

Fourier transform IR spectroscopy (FTIR) is used to analyze cells of Micrococcus luteus, the type species of the highly heterogeneous genus Micrococcus that belongs to the Micrococcaceae family. The cells of M. luteus, which is a Gram-positive and yellow-pigmented bacterium, are submitted to increasing doses of gamma radiation. Irradiation leads to the generation of reactive oxygen species that induce biochemical changes as shown in spectral profiles. Beyond a dose of 0.70 kGy, significant differences between samples are observed, particularly in the 1485-900 cm(-1) region, which contains information about membrane lipids, cell wall polysaccharides, and nucleic acids. After a dose of 16.50 kGy, M. luteus is reincubated for times ranging from 1 to 24 h. Postirradiation reincubated bacteria are found far from the control and irradiated cells (mainly in the 985-900 cm(-1) range), suggesting that a biomolecular rearrangement occurs as soon as reincubation begins in the growth medium. Thus, FTIR spectroscopy appears to be a very useful technique for the rapid visualization of the alterations induced by both the radiation and mutagenic response during reincubation. The use of mathematical methods gives good insight into the biomolecular compounds involved in these two mechanisms. In view of these preliminary results, we hypothesize that it can be successfully applied to any type of tissue and that it may be a future interesting tool for evaluating the effects of radiation in humans.