Identification of two possible metabolic pathways responsible for the biodegradation of 3, 5, 6-trichloro-2-pyridinol in Micrococcus luteus ML.

3, 5, 6-Trichloro-2-pyridinol (TCP) is a metabolite of the insecticide chlorpyrifos and the herbicide triclopyr, and it is higher toxic than the parent compounds. Microbially-mediated mineralization appears to be the primary degradative pathway and the important biological process of detoxification. However, little information is available on TCP complete metabolic pathways and mechanisms. In this study, the degradation of TCP was studied with a novel strain Micrococcus luteus ML isolated from a stable TCP degrading microbiota. Strain ML was capable of degrading 61.6% of TCP (50 mg/L) and 35.4% of chlorpyrifos (50 mg/L) at 24 h and 48 h under the optimal conditions (temperature: 35 °C; pH: 7.0), respectively. It could also degrade 3, 5-dichloro-2-pyridone, 6-chloropyridin-2-ol, 2-hydroxypyridine and phoxim when provided as sole carbon and energy sources. Seven TCP intermediate metabolites were detected in strain ML and two possible degradation pathways of TCP were proposed on the basis of LC-MS analysis. Both the hydrolytic-oxidative dechlorination pathway and the denitrification pathway might be involved in TCP biodegradation by strain ML. To the best of our knowledge, this is the first report on two different pathways responsible for TCP degradation in one strain, and this finding also provides novel information for studying the metabolic mechanism of TCP in pure culture.

Degradation and decolourization potential of ligninolytic enzyme producing Bacillus paramycoides BL2 and Micrococcus luteus BL3 for pulp paper industrial effluent and its toxicity evaluation.

Aim of this study was to optimize the production of Ligninolytic enzyme for the degradation of complex pollutants present in pulp paper industrial effluent (PPIE). Two ligninolytic enzyme-producing bacterial strains were isolated from PPIE and identified as Bacillus paramycoides strain BL2 (MZ676667) and Micrococcus luteus strains BL3 (MZ676668). The identified bacterial strain Bacillus paramycoides strain BL2 showed optimum production of LiP (4.30 U/ml), MnP (3.38 U/ml) at 72 h of incubation, while laccase (4.43 U/ml) at 96 h of incubation. While, Micrococcus luteus strains BL3 produced maximum LiP (3.98) and MnP (3.85 U/ml) at 96 h of incubation and maximum laccase (3.85 U/ml) at 72 h of incubation, pH 7-8, and temperatures of 30-35 °C. Furthermore, in the presence of glucose (1.0%) and peptone (0.5%) as nutrient sources, the enzyme activity of consortium leads to reduction of lignin (70%), colour (63%) along with COD (71%) and BOD (58%). The pollutants detected in control i.e. 3.6-Dioxa-2,7-disilaoctane, 2-Heptnoic acid,trimethylsilyl ester, 7-Methyldinaphtho [2,1-b,1',2'-d] silole, Hexadeconoic acid, trimethylysilyl ester, Methyl1(Z)-3,3-dipheny.1-4-hexenoale, 2,6,10,14,18,22-Tetracosahexane,2,2-dimethylpropyl(2Z,6E)-10,11epoxy5,6 Dihyrostigmasterol, acetate were completely diminished. The toxicity of PPIE was reduced up to 75%. Hence, knowledge of this study will be very useful for industrial sector for treatment of complex wastewater.

Micrococcus luteus strain CGK112 isolated from cow dung demonstrated efficient biofilm-forming ability and degradation potential toward high-density polyethylene (HDPE).

Biodegradation is the most promising environmentally sustainable method that offers a significant opportunity with minimal negative environmental consequences while searching for solutions to this global problem of plastic pollution that has now spread to almost everywhere in the entire world. In the present work, HDPE-degrading bacterial strain CGK112 was isolated from the fecal matter of a cow. The bacterial strain was identified as Micrococcus luteus CGK112 by 16S rRNA sequence coding analysis. Significant weight loss, i.e., 3.85% was recorded in the HDPE film treated with strain CGK112 for 90 days. The surface micromorphology was examined using FE-SEM, which revealed spectacular bacterial colonization as well as structural deformation. Furthermore, the EDX study indicated a significant decrease in the atomic percentage of carbon content, whereas FTIR analysis confirmed functional groups alternation as well as an increase in the carbonyl index which can be attributed to the metabolic activity of biofilm. Our findings provide insight into the capacity of our strain CGK112 to colonize and utilize HDPE as a single carbon source, thus promoting its degradation.

The Binding of Monoclonal and Polyclonal Anti-Z-DNA Antibodies to DNA of Various Species Origin.

DNA is a polymeric macromolecule that can display a variety of backbone conformations. While the classical B-DNA is a right-handed double helix, Z-DNA is a left-handed helix with a zig-zag orientation. The Z conformation depends upon the base sequence, base modification and supercoiling and is considered to be transient. To determine whether the presence of Z-DNA can be detected immunochemically, the binding of monoclonal and polyclonal anti-Z-DNA antibodies to a panel of natural DNA antigens was assessed by an ELISA using brominated poly(dG-dC) as a control for Z-DNA. As these studies showed, among natural DNA tested (Micrococcus luteus, calf thymus, Escherichiacoli, salmon sperm, lambda phage), micrococcal (MC) DNA showed the highest binding with both anti-Z-DNA preparations, and E. coli DNA showed binding with the monoclonal anti-DNA preparation. The specificity for Z-DNA conformation in MC DNA was demonstrated by an inhibition binding assay. An algorithm to identify propensity to form Z-DNA indicated that DNA from Mycobacterium tuberculosis could form Z-DNA, a prediction confirmed by immunoassay. Together, these findings indicate that anti-Z-DNA antibodies can serve as probes for the presence of Z-DNA in DNA of various species origin and that the content of Z-DNA varies significantly among DNA sources.

Phenotypic and genomic analysis of multiple heavy metal-resistant Micrococcus luteus strain AS2 isolated from industrial waste water and its potential use in arsenic bioremediation.

Multiple heavy metal-resistant bacterium, Micrococcus luteus strain AS2, was isolated from industrial waste water of District Sheikhupura, Pakistan. The isolated bacterium showed minimum inhibitory concentrations of 55 and 275 mM against arsenite and arsenate. The bacterial strain also showed resistance against other heavy metal ions, i.e., lead, cadmium, chromium, mercury, nickel, and zinc, apart from arsenic. The optimum temperature and pH were 37 °C and 7, respectively. The antioxidant enzymes such as catalase were significantly increased under arsenite stress. The increase in 43.9% of GSH/GSSG and 72.72% of non-protein thiol was determined under15 mM arsenite stress. Bacterial genome was sequenced through Illumina and Nanopore and genes related to arsenic and other heavy metals were identified and blast (tblastx) on NCBI. Through scanning electron microscopy, no morphological changes were observed in bacterial cells under arsenite stress. The peaks appeared in EDX showed that there is surface adsorption of arsenite in bacterial cell while it was confirmed from Fourier transformed infrared spectroscopy analysis that there is some interaction between arsenite and functional groups present on the surface of bacterial cell. The SDS-PAGE analysis of whole-cell proteins under 15 mM arsenite stress clearly revealed that there is upregulation of some proteins in ranged of 60 to 34 kDa. The bioremediation efficiency (E) of bacterial biomass was 72% after 2 h and 99% after 10 h. The bioremediation efficiency of bacterial biomass is an indicator for the isolated bacterium to employ as a potential candidate for the amelioration of sites contaminated with arsenic.

Bacterial Biotransformation of Oleic Acid: New Findings on the Formation of γ-Dodecalactone and 10-Ketostearic Acid in the Culture of Micrococcus luteus.

Microbial conversion of oleic acid (1) to form value-added industrial products has gained increasing scientific and economic interest. So far, the production of natural lactones with flavor and fragrance properties from fatty acids by non-genetically modified organisms (non-GMO) involves whole cells of bacteria catalyzing the hydration of unsaturated fatty acids as well as yeast strains responsible for further ß-oxidation processes. Development of a non-GMO process, involving a sole strain possessing both enzymatic activities, significantly lowers the costs of the process and constitutes a better method from the customers' point of view regarding biosafety issues. Twenty bacteria from the genus of Bacillus, Comamonas, Dietzia, Gordonia, Micrococcus, Pseudomonas, Rhodococcus and Streptomyces were screened for oxidative functionalization of oleic acid (1). Micrococcus luteus PCM525 was selected as the sole strain catalyzing the one-pot transformation of oleic acid (1) into natural valuable peach and strawberry-flavored γ-dodecalactone (6) used in the food, beverage, cosmetics and pharmaceutical industries. Based on the identified products formed during the process of biotransformation, we clearly established a pathway showing that oleic acid (1) is hydrated to 10-hydroxystearic acid (2), then oxidized to 10-ketostearic acid (3), giving 4-ketolauric acid (4) after three cycles of ß-oxidation, which is subsequently reduced and cyclized to γ-dodecalactone (6) (Scheme 1). Moreover, three other strains (Rhodococcus erythropolis DSM44534, Rhodococcus ruber PCM2166, Dietzia sp. DSM44016), with high concomitant activities of oleate hydratase and alcohol dehydrogenase, were identified as efficient producers of 10-ketostearic acid (3), which can be used in lubricant and detergent formulations. Considering the prevalence of γ-dodecalactone (6) and 10-ketostearic acid (3) applications and the economic benefits of sustainable management, microbial bioconversion of oleic acid (1) is an undeniably attractive approach.

Microbial degradation of low-density polyethylene and synthesis of polyhydroxyalkanoate polymers.

We have characterized the ability of eight bacterial strains to utilize powdered low-density polyethylene (LDPE) plastic (untreated and without any additives) as a sole carbon source. Cell mass production on LDPE-containing medium after 21 days of incubation varied between 0.083 ± 0.015 g/L cell dry mass (cdm) for Micrococcus luteus IRN20 and 0.39 ± 0.036 g/L for Cupriavidus necator H16. The percent decrease in LDPE mass ranged from 18.9% ± 0.72% for M. luteus IRN20 to 33.7% ± 1.2% for C. necator H16. Linear alkane hydrolysis products from LDPE degradation were detected in the culture media, and the carbon chain lengths of the hydrolysis products detected varied, depending on the species of bacteria. We also determined that C. necator H16 produced short-chain-length polyhydroxyalkanoate biopolymers, while Pseudomonas putida LS46 and Acinetobacter pittii IRN19 produced medium-chain-length biopolymers while growing on polyethylene powder. Cupriavidus necator H16 accumulated poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-V) polymers to 3.18% ± 0.4% of cdm. The monomer composition of the PHB-V was 94.9% ± 0.61% 3-hydroxybutyrate and 5.03% ± 0.56% 3-hydroxyvalerate. This is the first report that provides direct evidence for simultaneous bioconversion of LDPE plastic to biodegradable polyhydroxyalkanoate polymers.

Toxification of polycyclic aromatic hydrocarbons by commensal bacteria from human skin.

The ubiquitous occurrence of polycyclic aromatic hydrocarbons (PAHs) leads to constant human exposure at low levels. Toxicologically relevant are especially the high-molecular weight substances due to their (pro-)carcinogenic potential. Following ingestion or uptake, the eukaryotic phase I metabolism often activates these substances to become potent DNA binders, and unsurprisingly metabolism and DNA-adduct formation of model substances such as benzo[a]pyrene (B[a]P) are well studied. However, apart from being subjected to eukaryotic transformations PAHs are also carbon and energy sources for the myriads of commensal microbes inhabiting man's every surface. Yet, we know little about the microbiome's PAH-metabolism capacity and its potentially adverse impact on the human host. This study now shows that readily isolable skin commensals transform B[a]P into a range of highly cyto- and genotoxic metabolites that are excreted in toxicologically relevant concentrations during growth. The respective bacterial supernatants contain a mixture of established eukaryotic as well as hitherto unknown prokaryotic metabolites, the combination of which leads to an increased toxicity. Altogether we show that PAH metabolism of the microbiome has to be considered a potential hazard.

Isolation of Viable but Non-culturable Bacteria from Printing and Dyeing Wastewater Bioreactor Based on Resuscitation Promoting Factor.

Printing and dyeing wastewater with high content of organic matters, high colority, and poor biochemical performance is hard to be degraded. In this study, we isolated viable but non-culturable (VBNC) bacteria from printing and dyeing wastewater with the culture media contained resuscitation promoting factor (Rpf) protein secreted by Micrococcus luteus, counted the culturable cells number with the most probable number, sequenced 16S rRNA genes, and performed polymerase chain reaction-denaturing gradient gel electrophoresis. It is obviously that the addition of Rpf in the enrichment culture could promote growth and resuscitation of bacteria in VBNC state to obtain more fastidious bacteria significantly. The identified bacteria were assigned to nine genera in the treatment group, while the two strains of Ochrobactrum anthropi and Microbacterium sp. could not be isolated from the control group. The function of isolated strains was explored and these strains could degrade the dye of Congo red. This study provides a new sight into the further study including the present state, composition, formation mechanism, and recovery mechanism about VBNC bacteria in printing and dyeing wastewater, which would promote to understand bacterial community in printing and dyeing wastewater, and to obtain VBNC bacteria from ecological environment.

Radiation-Resistant Micrococcus luteus SC1204 and Its Proteomics Change Upon Gamma Irradiation.

To explore the radiation-resistance mechanisms in bacteria, a radiation-resistant strain SC1204 was isolated from the surrounding area of a (60)Co-γ radiation facility. SC1204 could survive up to 8 kGy dose of gamma irradiation and was identified as Micrococcus luteus by phylogenetic analysis of 16S rRNA gene sequences. Its proteomic changes under 2-kGy irradiation were examined by two-dimensional electrophoresis followed by MALDI-TOF-TOF/MS analysis. The results showed that at least 24 proteins displayed significant changes (p < 0.05) at expression level under the radiation stress, among which 22 were successfully identified and classified into the major functional categories of metabolism, energy production and conservation, translation, ribosomal structure, and biogenesis. Among these proteins, leucyl aminopeptidase involved in synthesis of glutathione was the most abundant induced protein during postirradiation recovery, indicating that anti-oxidation protection was the most important line of defense in SC1204 against radiation. The next abundant protein was phosphoribosyl aminoimidazole carboxamide formyltransferase/IMP cyclohydrolase (AICAR Tfase/IMPCH), the key enzyme in the biosynthetic pathway of purine that is anti-radiation compound. Other proteins changing significantly (p < 0.05) after radiation exposure included urocanate hydratase, dihydrolipoyl dehydrogenase, succinyl-CoA synthetase subunit alpha, phosphoglycerate kinase, cell division protein FtsZ, elongation factor Ts and Tu, translation elongation factor Tu and G, 30S ribosomal protein S1, histidyl-tRNA synthetase, and arginyl-tRNA synthetase, which were considered to be the key proteins in urocanate metabolism, tricarboxylic acid cycle, glycolysis, cell division process, and synthesis process of proteins. Therefore, these proteins may also play important roles in radiation resistance in M. luteus.

Phytoremediation of fuel oil and lead co-contaminated soil by Chromolaena odorata in association with Micrococcus luteus.

Phytoremediation is widely promoted as a cost-effective technology for treating heavy metal and total petroleum hydrocarbon (TPH) co-contaminated soil. This study investigated the concurrent removal of TPHs and Pb in co-contaminated soil (27,000 mg kg(-1) TPHs, 780 mg kg(-1) Pb) by growing Siam weed (Chromolaena odorata) in a pot experiment for 90 days. There were four treatments: co-contaminated soil; co-contaminated soil with C. odorata only; co-contaminated soil with C. odorata and Micrococcus luteus inoculum; and co-contaminated soil with M. luteus only. C. odorata survived and grew well in the co-contaminated soil. C. odorata with M. luteus showed the highest Pb accumulation (513.7 mg kg(-1)) and uptake (7.7 mg plant(-1)), and the highest reduction percentage of TPHs (52.2%). The higher TPH degradation in vegetated soils indicated the interaction between the rhizosphere microorganisms and plants. The results suggested that C. odorata together with M. luteus and other rhizosphere microorganisms is a promising candidate for the removal of Pb and TPHs in co-contaminated soils.

Reclamation of DPK hydrocarbon polluted agricultural soil using a selected bulking agent.

In the present study, laboratory scale bioremediation of dual purpose kerosene (DPK) hydrocarbon polluted soil using bulking agent (saw dust) was carried out. The effect of different parameters such as total petroleum hydrocarbon (TPH), dehydrogenase activity (DHase) and pH on bioremediation performance were evaluated. Studied parameters such as microbial dynamics, percentage degradation (95.20%), DHase (8.20 ± 0.43) were found to be higher in saw dust amended system and significantly differed with control at p < 0.05. Experimental data adequately fitted the first order kinetic thus, generated r(2) values (0.966), first order degradation constant (0.659 d(-1)), and degradation half-life t1/2 = ln2/k (1.05 d). Micrococcus luteus, Bacillus sp., Rhizopus arrhizus and Aspergillus sp. were isolated from the study. The use of saw dust as bulking agent greatly increased biodegradation rate and resulted in effective DPK hydrocarbon clean up. Therefore, saw dust could serve as an effective biostimulant towards improved bioremediation of hydrocarbon polluted environment.

Action of the multifunctional peptide BP100 on native biomembranes examined by solid-state NMR.

Membrane composition is a key factor that regulates the destructive activity of antimicrobial peptides and the non-leaky permeation of cell penetrating peptides in vivo. Hence, the choice of model membrane is a crucial aspect in NMR studies and should reflect the biological situation as closely as possible. Here, we explore the structure and dynamics of the short multifunctional peptide BP100 using a multinuclear solid-state NMR approach. The membrane alignment and mobility of this 11 amino acid peptide was studied in various synthetic lipid bilayers with different net charge, fluidity, and thickness, as well as in native biomembranes harvested from prokaryotic and eukaryotic cells. (19)F-NMR provided the high sensitivity and lack of natural abundance background that are necessary to observe a labelled peptide even in protoplast membranes from Micrococcus luteus and in erythrocyte ghosts. Six selectively (19)F-labeled BP100 analogues gave remarkably similar spectra in all of the macroscopically oriented membrane systems, which were studied under quasi-native conditions of ambient temperature and full hydration. This similarity suggests that BP100 has the same surface-bound helical structure and high mobility in the different biomembranes and model membranes alike, independent of charge, thickness or cholesterol content of the system. (31)P-NMR spectra of the phospholipid components did not indicate any bilayer perturbation, so the formation of toroidal wormholes or micellarization can be excluded as a mechanism of its antimicrobial or cell penetrating action. However, (2)H-NMR analysis of the acyl chain order parameter profiles showed that BP100 leads to considerable membrane thinning and thereby local destabilization.

Microbial production of aliphatic (S)-epoxyalkanes by using Rhodococcus sp. strain ST-10 styrene monooxygenase expressed in organic-solvent-tolerant Kocuria rhizophila DC2201.

We describe the development of biocatalysis for producing optically pure straight-chain (S)-epoxyalkanes using styrene monooxygenase of Rhodococcus sp. strain ST-10 (RhSMO). RhSMO was expressed in the organic solvent-tolerant microorganism Kocuria rhizophila DC2201, and the bioconversion reaction was performed in an organic solvent-water biphasic reaction system. The biocatalytic process enantioselectively converted linear terminal alkenes to their corresponding (S)-epoxyalkanes using glucose and molecular oxygen. When 1-heptene and 6-chloro-1-hexene were used as substrates (400 mM) under optimized conditions, 88.3 mM (S)-1,2-epoxyheptane and 246.5 mM (S)-1,2-epoxy-6-chlorohexane, respectively, accumulated in the organic phase with good enantiomeric excess (ee; 84.2 and 95.5%). The biocatalysis showed broad substrate specificity toward various aliphatic alkenes, including functionalized and unfunctionalized alkenes, with good to excellent ee. Here, we demonstrate that this biocatalytic system is environmentally friendly and useful for producing various enantiopure (S)-epoxyalkanes.

Physicochemical properties and membrane biofouling of extra-cellular polysaccharide produced by a Micrococcus luteus strain.

The physicochemical properties of the extra-cellular polysaccharide (EPS) produced by a Micrococcus luteus strain, a dominating strain isolated from membrane biofouling layer, were determined in this study. The EPS isolated from this strain was measured to have an average molecular weight of 63,540 Da and some typical polysaccharide absorption peaks in Fourier transform infrared spectrum. Monosaccharide components of the EPS contained rhamnose, fucose, arabinose, xylose, mannose, galactose and glucose in a molar ratio of 0.2074:0.0454:0.0262:0.0446:1.7942:1.2086:0.4578. Pseudo plastic properties were also observed for the EPS through the rheological measurement. The EPS was further characterized for its behavior to cause membrane flux decline. The results showed that both flux declines for polyvinylidenefluoride (PVDF) and polypropylene membranes became more severe as EPS feed concentration increased. A higher irreversible fouling for the PVDF membrane suggested that the EPS had the larger fouling potential to this microfiltration membrane.

Micrococcus luteus-derived extracellular vesicles attenuate neutrophilic asthma by regulating miRNAs in airway epithelial cells.

Bacterial extracellular vesicles (EVs) have been shown to regulate various pulmonary diseases, but their functions in asthma remain uncertain. To demonstrate the clinical significance of Micrococcus luteus-derived EVs (MlEVs) in asthma, we enrolled 45 asthmatic patients (20 patients with neutrophilic asthma [NA], 25 patients with eosinophilic asthma [EA]) and 40 healthy controls (HCs). When the prevalence of IgG1 and IgG4 specific to MlEVs was evaluated in serum by ELISA, lower levels of MlEV-specific IgG4 (but not IgG1) were noted in asthmatic patients than in HCs. Among asthmatic patients, significantly lower levels of MIEV-specific IgG4 were noted in patients with NA than in those with EA. Moreover, there was a positive correlation between serum MlEV-specific IgG4 levels and FEV1 (%) values. In asthmatic C57BL/6 mice, MlEVs significantly attenuated neutrophilic airway inflammation by reducing the production of IL-1ß and IL-17 in bronchoalveolar lavage fluid as well as the number of group 3 innate lymphoid cells (ILC3s) in lung tissues. To clarify the functional mechanism of MlEVs in NA, the effect of MlEVs on airway epithelial cells (AECs) and immune cells was investigated ex vivo. According to microarray analysis, MlEVs upregulated hsa-miR-4517 expression in AECs. Moreover, this miRNA could suppress IL-1ß production by monocytes, resulting in the inhibition of ILC3 activation and neutrophil recruitment. These findings suggest that MlEVs could be a novel therapeutic agent for managing unresolved NA by regulating miRNA expression in AECs.

Structure of FabH and factors affecting the distribution of branched fatty acids in Micrococcus luteus.

Micrococcus luteus is a Gram-positive bacterium that produces iso- and anteiso-branched alkenes by the head-to-head condensation of fatty-acid thioesters [coenzyme A (CoA) or acyl carrier protein (ACP)]; this activity is of interest for the production of advanced biofuels. In an effort to better understand the control of the formation of branched fatty acids in M. luteus, the structure of FabH (MlFabH) was determined. FabH, or ß-ketoacyl-ACP synthase III, catalyzes the initial step of fatty-acid biosynthesis: the condensation of malonyl-ACP with an acyl-CoA. Analysis of the MlFabH structure provides insights into its substrate selectivity with regard to length and branching of the acyl-CoA. The most structurally divergent region of FabH is the L9 loop region located at the dimer interface, which is involved in the formation of the acyl-binding channel and thus limits the substrate-channel size. The residue Phe336, which is positioned near the catalytic triad, appears to play a major role in branched-substrate selectivity. In addition to structural studies of MlFabH, transcriptional studies of M. luteus were also performed, focusing on the increase in the ratio of anteiso:iso-branched alkenes that was observed during the transition from early to late stationary phase. Gene-expression microarray analysis identified two genes involved in leucine and isoleucine metabolism that may explain this transition.

Bacteriolytic activity of human interleukin-2.

In this paper we report the discovery of bacteriolytic activity of an immune system cytokine mediator, interleukin-2. Bacteriolytic activity of interleukin-2 was compared with a well-known bacteriolytic enzyme - chicken egg white lysozyme - by monitoring the lysis of the Gram-negative bacterium Escherichia coli, the Gram-positive coccus Micrococcus luteus, and the Gram-positive spore-forming bacillus Bacillus subtilis. It was found that interleukin-2 has greater specificity to the Gram-negative bacterium E. coli than does lysozyme. In contrast to chicken egg white lysozyme, interleukin-2 does not lyse the Gram-positive coccus M. luteus and the Gram-positive spore-forming bacillus B. subtilis. These results give a new understanding of the biological functions of interleukin-2, a regulatory protein that plays a role in oncological and infectious diseases.

Viability and biomass of Micrococcus luteus DE2008 at different salinity concentrations determined by specific fluorochromes and CLSM-image analysis.

In previous studies, our group developed a method based on Confocal Laser Scanning Microscopy and Image Analysis (CLSM-IA) to analyze the diversity and biomass of cyanobacteria in microbial mats. However, this method cannot be applied to heterotrophic microorganisms, as these do not have autofluorescence. In this article, we present a method that combines CLSM-IA and Hoechst 33342 and SYTOX Green fluorochromes (FLU-CLSM-IA) to determine the viability and biomass of Micrococcus luteus DE2008, isolated from a saline microbial mat (Ebro Delta, Tarragona, Spain). The method has been applied to assess the effect of salinity on this microorganism. A reduction in viability and biomass (live cells) was observed as the salt concentration increases. The largest effect was at 100‰ NaCl with a cell death of 27.25% and a decrease in total and individual biomass of 39.75 and 0.009 mgC/cm(3), respectively, both with respect to optimal growth (10 ‰ NaCl). On the other hand, another important contribution of this article was that combining the FLU-CLSM-IA results with those achieved by plate counts enabled us to determine, for first time, the viability and the total biomass of the "dormant cells" (66.75% of viability and 40.59 mgC/cm(3) of total biomass at 100‰ NaCl). FLU-CLSM-IA is an efficient, fast, and reliable method for making a total count of cells at pixel level, including the dormant cells, to evaluate the viability and the biomass of a hetetrophic microorganism, M. luteus DE2008.

[Influence of stressors on formation of uncultured microorganism forms and their recovery].

Data of the last decade on the studies of influence of various inductors of microorganism uncultured state and reasons of reversion that include not only effect of appropriate cultivation conditions but also simple cancellation of unfavorable effects are analyzed. Reasons for transition to uncultured state are discussed; those could be a variety of factors such as heat, alkaline, acid and osmotic stress. Factors that promote recovery from uncultured state with the main being Rpf (Resustication promoting factor) factor isolated from Micrococcus luteus and cancellation of endogenic peroxide effect by addition of catalase and sodium pyruvate are analyzed.