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Background: Built environments (BEs) are typically considered to be oligotrophic and harsh environments for microbial communities under normal, non-damp conditions. However, the metabolic functions of microbial inhabitants in BEs remain poorly understood. This study aimed to shed light on the functional capabilities of microbes in BEs by analyzing 860 representative metagenome-assembled genomes (rMAGs) reconstructed from 738 samples collected from BEs across the city of Hong Kong and from the skin surfaces of human occupants. The study specifically focused on the metabolic functions of rMAGs that are either phylogenetically novel or prevalent in BEs., Results: The diversity and composition of BE microbiomes were primarily shaped by the sample type, with Micrococcus luteus and Cutibacterium acnes being prevalent. The metabolic functions of rMAGs varied significantly based on taxonomy, even at the strain level. A novel strain affiliated with the Candidatus class Xenobia in the Candidatus phylum Eremiobacterota and two novel strains affiliated with the superphylum Patescibacteria exhibited unique functions compared with their close relatives, potentially aiding their survival in BEs and on human skins. The novel strains in the class Xenobia possessed genes for transporting nitrate and nitrite as nitrogen sources and nitrosative stress mitigation induced by nitric oxide during denitrification. The two novel Patescibacteria strains both possessed a broad array of genes for amino acid and trace element transport, while one of them carried genes for carotenoid and ubiquinone biosynthesis. The globally prevalent M. luteus in BEs displayed a large and open pangenome, with high infraspecific genomic diversity contributed by 11 conspecific strains recovered from BEs in a single geographic region. The versatile metabolic functions encoded in the large accessory genomes of M. luteus may contribute to its global ubiquity and specialization in BEs., Conclusions: This study illustrates that the microbial inhabitants of BEs possess metabolic potentials that enable them to tolerate and counter different biotic and abiotic conditions. Additionally, these microbes can efficiently utilize various limited residual resources from occupant activities, potentially enhancing their survival and persistence within BEs. A better understanding of the metabolic functions of BE microbes will ultimately facilitate the development of strategies to create a healthy indoor microbiome. Video Abstract., (© 2024. The Author(s).)

The viable but non-culturable (VBNC) is an inactive state, and certain bacteria can enter under adverse conditions. The VBNC state challenges the environment, food safety, and public health since VBNCs may resuscitate and pose a risk to human health. The aim of this study was to investigate the effect of plasma-generated nitric oxide water (PG-NOW) on airborne contaminant Micrococcus luteus (M. luteus) and examine its potential to induce the VBNC state. The essential conditions for bacteria to enter VBNC state are low metabolic activity and rare or no culturable counts. The results indicated that PG-NOW effectively eliminates M. luteus, and the remaining bacteria are in culturable condition. Moreover, the conventional cultured-based method combined with a propidium iodide monoazide quantitative PCR (PMAxx TM -qPCR) showed no significant VBNC induction and moderate culturable counts. Results from the qPCR revealed that gene levels in PG-NOW treated bacteria related to resuscitation-promoting factors, amino acid biosynthesis, and fatty acid metabolism were notably upregulated. PG-NOW inactivated M. luteus showed negligible VBNC formation and alleviated infection ability in lung cells. This study provides new insights into the potential use of PG-NOW reactive species for the prevention and control of the VBNC state of M. luteus., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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 FEV 1 (%) values. In asthmatic C57BL/6 mice, MlEVs significantly attenuated neutrophilic airway inflammation by reducing the production of IL-1β 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-1β 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., (© 2023. The Author(s).)

Mutants of Micrococcus luteus strain ATCC49732 lacking the yellow pigment sarcinaxanthin were observed at an unexpectedly high frequency and the molecular basis was investigated. PCR probing revealed complete deletion of the crt biosynthetic operon in 11/14 mutants. Inverse PCR was used to identify a common breakpoint 35 kb downstream from crt precisely at the end of the right inverted repeat (IRR) of a partial ISMlu8 element that lies between two inversely oriented full-length ISMlu2. A total of three different breakpoints 5' to crt were found with the sequence CTAG one bp 5' to each novel junction. Analysis of 35 genomic sites with single ISMlu8 insertions showed that ISMlu8 transposase has high specificity for CTAG, implicating its key role in formation of the Δcrt deletions. No downstream deletion endpoints were observed at an immediately adjacent ISMlu8 with a nearly identical IRR in the same orientation and slightly closer to the crt operon, indicating that access of ISMlu8 transposase to the ISMlu2-flanked ISMlu8 IRR is greatly enhanced by the surrounding oppositely oriented ISMlu2s. The association of high frequency genomic rearrangement with this distinctive natural configuration of ISs from two different IS families offers a new insight into IS element evolutionary potential., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

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., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Abs to DNA (anti-DNA) are a unique population of Abs that bind structural determinants on the DNA molecule. In systemic lupus erythematosus (SLE), anti-DNA Abs bind to conserved antigenic determinants, with the phosphodiester backbone being the most likely. In contrast, otherwise healthy subjects (HS) express anti-DNA that bind selectively to nonconserved sites on certain bacterial and viral DNA. As shown previously, SLE anti-DNA bind by a mechanism termed Fc-dependent monogamous bivalency. In this mechanism, both Fab sites interact with determinants on the same extended DNA molecule, reflecting the low affinity of each Fab site; the requirement for the Fc region suggests some contribution of the C region to increase avidity. In this study, we investigated whether anti-DNA from HS also bind to bacterial DNA by Fc-dependent monogamous bivalency. For this purpose, we compared the activity of intact IgG with Fab and F(ab') 2 fragments prepared from the plasmas of SLE patients and HS using ELISAs with DNA from calf thymus or Micrococcus luteus These studies showed that Fab fragments from all plasmas tested, both SLE and HS, failed to bind significantly to DNA compared with intact IgG. By contrast, some, but not all, F(ab') 2 preparations from both SLE patients and HS showed binding to M. luteus DNA; F(ab') 2 fragments from SLE plasmas, however, did not bind significantly to calf thymus DNA. Together, these findings suggest that although anti-DNA Abs, whether from SLE or HS, bind by monogamous bivalency, binding to bacterial DNA does not require the Fc region., (Copyright © 2021 The Authors.)

Historically, Micrococcus luteus was one of the first organisms used to study natural transformation, one of the main routes of horizontal gene transfer among prokaryotes. However, little is known about the molecular basis of competence development in M. luteus or any other representative of the phylum of high-GC Gram-positive bacteria (Actinobacteria), while this means of genetic exchange has been studied in great detail in Gram-negative and low-GC Gram-positive bacteria (Firmicutes). In order to identify new genetic elements involved in regulation of the comEA - comEC competence operon in M. luteus , we conducted random chemical mutagenesis of a reporter strain expressing lacZ under the control of the comEA - comEC promoter, followed by the screening of dysregulated mutants. Mutants with (i) upregulated com promoter under competence-repressing conditions and (ii) mutants with a repressed com promoter under competence-inducing conditions were isolated. After genotype and phenotype screening, the genomes of several mutant strains were sequenced. A selection of putative com -influencing mutations was reinserted into the genome of the M. luteus reporter strain as markerless single-nucleotide mutations to confirm their effect on com gene expression. This strategy revealed mutations affecting com gene expression at genetic loci different from previously known genes involved in natural transformation. Several of these mutations decreased transformation frequencies by several orders of magnitude, thus indicating significant roles in competence development or DNA acquisition in M. luteus . Among the identified loci, there was a new locus containing genes with similarity to genes of the tad clusters of M. luteus and other bacteria .

Background: Micrococcus luteus is a group of actinobacteria that is widely used in biotechnology and is being thought as an emerging nosocomial pathogen. With one of the smallest genomes of free-living actinobacteria, it is found in a wide range of environments, but intraspecies genetic diversity and adaptation strategies to various environments remain unclear. Here, comparative genomics, phylogenomics, and genome-wide association studies were used to investigate the genomic diversity, evolutionary history, and the potential ecological differentiation of the species., Results: High-quality genomes of 66 M. luteus strains were downloaded from the NCBI GenBank database and core and pan-genome analysis revealed a considerable intraspecies heterogeneity. Phylogenomic analysis, gene content comparison, and average nucleotide identity calculation consistently indicated that the species has diverged into three well-differentiated clades. Population structure analysis further suggested the existence of an unknown ancestor or the fourth, yet unsampled, clade. Reconstruction of gene gain/loss events along the evolutionary history revealed both early events that contributed to the inter-clade divergence and recent events leading to the intra-clade diversity. We also found convincing evidence that recombination has played a key role in the evolutionary process of the species, with upto two-thirds of the core genes having been affected by recombination. Furthermore, distribution of mammal-associated strains (including pathogens) on the phylogenetic tree suggested that the last common ancestor had a free-living lifestyle, and a few recently diverged lineages have developed a mammal-associated lifestyle separately. Consistently, genome-wide association analysis revealed that mammal-associated strains from different lineages shared genes functionally relevant to the host-associated lifestyle, indicating a recent ecological adaption to the new host-associated habitats., Conclusions: These results revealed high intraspecies genomic diversity of M. luteus and highlighted that gene gain/loss events and extensive recombination events played key roles in the genome evolution. Our study also indicated that, as a free-living species, some lineages have recently developed or are developing a mammal-associated lifestyle. This study provides insights into the mechanisms that drive the genome evolution and adaption to various environments of a bacterial species.

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.

Natural competence for genetic transformation refers to the natural ability of various bacteria to take up exogenous DNA from their surroundings and to incorporate internalized genetic information into their genomes. By promoting bacterial diversification and adaptability, this process represents a major driving force in bacterial evolution. Micrococcus luteus was one of the first organisms used to study natural transformation in bacteria. Since then, however, only very little information about this phenomenon has been reported in M. luteus or in any member of the Actinobacteria phylum (low-GC Gram-positive bacteria). Previous work in our group indicated major differences between the transformation apparatus of M. luteus and the transformation machinery described for various Gram-negative and Gram-positive model bacteria belonging to the phyla Proteobacteria and Firmicutes (high-GC Gram-positive bacteria). This prompted us to initiate a study concerning the regulation mechanism of competence development in M. luteus. In this report, we identify amino acids as a nutritional factor that influences competence in a concentration-dependent manner. By using a transcriptional reporter strain for one of the late competence genes, we demonstrate how increasing concentrations of both amino acids mixtures and single amino acids supplemented to the growth medium affect transformability on transcriptional and post-transcriptional level. Furthermore, we revisit previously generated auxotrophic mutants to show that the transformation machinery is turned down during a state of extreme hunger for amino acids presumably as a part of a general response to auxotrophy. Finally, by generating and analysing knockout mutants for two predicted stringent response enzymes, we provide evidence for the involvement of the alarmone (p)ppGpp as a putative mediator of the effects on transformation development caused by amino acids. As a member of the Actinobacteria phylum, M. luteus could serve as a model for other representatives of the phylum, including a number of important human pathogens.

Both animals and amoebae use phagocytosis and DNA-based extracellular traps as anti-bacterial defense mechanisms. Whether, like animals, amoebae also use tissue-level barriers to reduce direct contact with bacteria has remained unclear. We have explored this question in the social amoeba Dictyostelium discoideum , which forms plaques on lawns of bacteria that expand as amoebae divide and bacteria are consumed. We show that CadA, a cell adhesion protein that functions in D. discoideum development, is also a bacterial agglutinin that forms a protective interface at the plaque edge that limits exposure of vegetative amoebae to bacteria. This interface is important for amoebal survival when bacteria-to-amoebae ratios are high, optimizing amoebal feeding behavior, and protecting amoebae from oxidative stress. Lectins also control bacterial access to the gut epithelium of mammals to limit inflammatory processes; thus, this strategy of antibacterial defense is shared across a broad spectrum of eukaryotic taxa.

Competition assays measure differences between populations of bacteria after stress adaptation, populations of different bacteria and mutations in antibiotic resistance genes. We have developed a competition-based assay to evaluate if genes upregulated under starvation are important for bacterial survival. Stress responses are critical for survival in non-pathogenic and pathogenic bacteria alike including Mycobacterium tuberculosis, Enterococcus fecaelis, Escherichia coli and Staphylococcus aureus. Unfortunately, most stress-survival proteins are poorly understood because suitable model bacteria and techniques are limited. To address this problem, we have engineered Micrococcus luteus NCTC 2665 (M. luteus) for competition assays by inactivating the sarcinaxanthin biosynthesis gene crtE (ΔcrtE), changing M. luteus colonies from yellow to white. This change allows easy identification in mixed cultures. The crtE knockout is relatively neutral for growth in complex and minimal acetate media and shows a measured fitness of one in competition with yellow wild-type bacteria. The ΔcrtE M. luteus competition assay identified a competition defect in a M. luteus strain when a specific universal stress protein was inactivated, suggesting a negative survival phenotype for this protein. We anticipate this competition assay can identify defects in other gene knockouts and mutational studies in M. luteus and will enhance our understanding of bacterial survival mechanisms., (© FEMS 2019.)

Acute hepatopancreatic necrosis disease (AHPND) was first reported in China in 2009 and afterwards in Mexico in 2013. AHPND is caused by Vibrio parahaemolyticus and affects Penaeus monodon and Litopenaeus vannamei shrimp cultures. The bacterium contains the pirA- and pirB-like genes in 69- to 70-Kb plasmids, which encode the toxins that produce the disease. The aim of this study was to determine whether pirA- and pirB-like genes existed in bacterial genera distinct from Vibrio before the first cases of AHPND were documented in Mexico. Two bacterial isolates were selected from shrimp farms in Nayarit in 2006 and analysed by nested-PCR to determine the presence of pirA- and pirB-like genes. The two isolates chosen did indeed show the presence of these genes, and those findings were confirmed by sequencing. Both strains matched to the bacterial species Micrococcus luteus. Results revealed two important situations: (a) the pirA- and pirB-like genes were present in a bacterial species that has not been reported previously (Micrococcus luteus); and (b) pirA- and pirB-like bacterial genes were present in Mexico before the first AHPND outbreak was reported in China., (© 2018 John Wiley & Sons Ltd.)

Mitomycin C (MC), an antitumor drug, and decarbamoylmitomycin C (DMC), a derivative of MC, alkylate DNA and form deoxyguanosine monoadducts and interstrand crosslinks (ICLs). Interestingly, in mammalian culture cells, MC forms primarily deoxyguanosine adducts with a 1"-R stereochemistry at the guanine-mitosene bond (1"-α) whereas DMC forms mainly adducts with a 1"-S stereochemistry (1"-β). The molecular basis for the stereochemical configuration exhibited by DMC has been investigated using biomimetic synthesis. Here, we present the results of our studies on the monoalkylation of DNA by DMC. We show that the formation of 1"-β-deoxyguanosine adducts requires bifunctional reductive activation of DMC, and that monofunctional activation only produces 1"-α-adducts. The stereochemistry of the deoxyguanosine adducts formed is also dependent on the regioselectivity of DNA alkylation and on the overall DNA CG content. Additionally, we found that temperature plays a determinant role in the regioselectivity of duplex DNA alkylation by mitomycins: At 0 °C, both deoxyadenosine (dA) and deoxyguanosine (dG) alkylation occur whereas at 37 °C, mitomycins alkylate dG preferentially. The new reaction protocols developed in our laboratory to investigate DMC-DNA alkylation raise the possibility that oligonucleotides containing DMC 1"-β-deoxyguanosine adducts at a specific site may be synthesized by a biomimetic approach., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Dormancy is a protective state in which diverse bacteria, including Mycobacterium tuberculosis , Staphylococcus aureus , Treponema pallidum (syphilis), and Borrelia burgdorferi (Lyme disease), curtail metabolic activity to survive external stresses, including antibiotics. Evidence suggests dormancy consists of a continuum of interrelated states, including viable but nonculturable (VBNC) and persistence states. VBNC and persistence contribute to antibiotic tolerance, reemergence from latent infections, and even quorum sensing and biofilm formation. Previous studies indicate that the protein mechanisms regulating persistence and VBNC states are not well understood. We have queried the VBNC state of Micrococcus luteus NCTC 2665 (MI-2665) by quantitative proteomics combining gel electrophoresis, high-performance liquid chromatography, and tandem mass spectrometry to elucidate some of these mechanisms. MI-2665 is a nonpathogenic actinobacterium containing a small (2.5-Mb), high-GC-content genome which exhibits a well-defined VBNC state induced by nutrient deprivation. The MI-2665 VBNC state demonstrated a loss of protein diversity accompanied by increased levels of 18 proteins that are conserved across actinobacteria, 14 of which have not been previously identified in VNBC. These proteins implicate an anaplerotic strategy in the transition to VBNC, including changes in the glyoxylate shunt, redox and amino acid metabolism, and ribosomal regulatory processes. Our data suggest that MI-2665 is a viable model for dissecting the protein mechanisms underlying the VBNC stress response and provide the first protein-level signature of this state. We expect that this protein signature will enable future studies deciphering the protein mechanisms of dormancy and identify novel therapeutic strategies effective against antibiotic-tolerant bacterial infections. IMPORTANCE Dormancy is a protective state enabling bacteria to survive antibiotics, starvation, and the immune system. Dormancy is comprised of different states, including persistent and viable but nonculturable (VBNC) states that contribute to the spread of bacterial infections. Therefore, it is imperative to identify how bacteria utilize these different dormancy states to survive antibiotic treatment. The objective of our research is to eliminate dormancy as a route to antibiotic tolerance by understanding the proteins that control dormancy in Micrococcus luteus NCTC 2665. This bacterium has unique advantages for studying dormancy, including a small genome and a well-defined and reproducible VBNC state. Our experiments implicate four previously identified and 14 novel proteins upregulated in VBNC that may regulate this critical survival mechanism., (Copyright © 2017 American Society for Microbiology.)

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.

The first β-lactone synthetase enzyme is reported, creating an unexpected link between the biosynthesis of olefinic hydrocarbons and highly functionalized natural products. The enzyme OleC, involved in the microbial biosynthesis of long-chain olefinic hydrocarbons, reacts with syn- and anti-β-hydroxy acid substrates to yield cis- and trans-β-lactones, respectively. Protein sequence comparisons reveal that enzymes homologous to OleC are encoded in natural product gene clusters that generate β-lactone rings, suggesting a common mechanism of biosynthesis.

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.

Enzyme fusion was investigated as a strategy to improve productivity of a two-step whole-cell biocatalysis. The biotransformation of long-chain sec-alcohols into esters by an alcohol dehydrogenase (ADH) and Baeyer-Villiger monooxygenases (BVMOs) was used as the model reaction. The recombinant Escherichia coli, expressing the fusion enzymes between the ADH of Micrococcus luteus NCTC2665 and the BVMO of Pseudomonas putida KT2440 or Rhodococcus jostii RHA1, showed significantly greater bioconversion activity with long-chain sec-alcohols (e.g., 12-hydroxyoctadec-9-enoic acid (1a), 13-hydroxyoctadec-9-enoic acid (2a), 14-hydroxyicos-11-enoic acid (4a)) when compared to the recombinant E. coli expressing the ADH and BVMOs independently. For instance, activity of the recombinant E. coli expressing the ADH-Gly-BVMO, in which glycine-rich peptide was used as the linker, with 1a was increased up to 22 μmol g dry cells(-1) min(-1). This value is over 40 % greater than the recombinant E. coli expressing the ADH and BVMO independently. The substantial improvement appeared to be driven by an increase in the functional expression of the BVMOs and/or an increase in mass transport efficiency by localizing two active sites in close proximity.

Here we report recombinant expression and activity of several type I fatty acid synthases that can function in parallel with the native Escherichia coli fatty acid synthase. Corynebacterium glutamicum FAS1A was the most active in E. coli and this fatty acid synthase was leveraged to produce oleochemicals including fatty alcohols and methyl ketones. Coexpression of FAS1A with the ACP/CoA-reductase Maqu2220 from Marinobacter aquaeolei shifted the chain length distribution of fatty alcohols produced. Coexpression of FAS1A with FadM, FadB, and an acyl-CoA-oxidase from Micrococcus luteus resulted in the production of methyl ketones, although at a lower level than cells using the native FAS. This work, to our knowledge, is the first example of in vivo function of a heterologous fatty acid synthase in E. coli. Using FAS1 enzymes for oleochemical production have several potential advantages, and further optimization of this system could lead to strains with more efficient conversion to desired products. Finally, functional expression of these large enzyme complexes in E. coli will enable their study without culturing the native organisms., (Published by Elsevier Inc.)

We demonstrated for the first time that the archaeal chaperones (i.e., γ-prefoldin and thermosome) can stabilize enzyme activity in vivo. Ricinoleic acid biotransformation activity of recombinant Escherichia coli expressing Micrococcus luteus alcohol dehydrogenase and the Pseudomonas putida KT2440 Baeyer-Villiger monooxygenase improved significantly with co-expression of γ-prefoldin or recombinant themosome originating from the deep-sea hyperthermophile archaea Methanocaldococcus jannaschii. Furthermore, the degree of enhanced activity was dependent on the expression levels of the chaperones. For example, whole-cell biotransformation activity was highest at 12 µmol/g dry cells/min when γ-prefoldin expression level was approximately 46% of the theoretical maximum. This value was approximately two-fold greater than that in E. coli, where the γ-prefoldin expression level was zero or set to the theoretical maximum. Therefore, it was assumed that the expression levels of chaperones must be optimized to achieve maximum biotransformation activity in whole-cell biocatalysts., (© 2014 Wiley Periodicals, Inc.)

Objective: To produce 10-ketostearic acid from oleic acid., Results: Oleic acid was converted to 10-ketostearic acid by a recombinant Corynebacterium glutamicum ATCC 13032 expressing oleate hydratase from Stenotrophomonas maltophilia and a secondary alcohol dehydrogenase from Micrococcus luteus under the control of a synthetic constitutive promoter. Optimal conditions for 10-ketostearic acid production were pH 7.5 and 30 °C with 5 g cells l(-1) and 2.5 g oleic acid l(-1). Under these conditions, the cells produced 1.96 g 10-ketostearic acid l(-1) from oleic acid in 6 h, with a conversion yield of 78 % (w) and a maximum volumetric productivity of 1.67 g l(-1) h(-1)., Conclusion: This is the first report of 10-ketostearic acid production using a recombinant C. glutamicum.

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., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Phylogenetic analysis of aerobic chemoorganotrophic bacteria of the two extreme regions (Dead Sea and West Antarctic) was performed on the basis of the nucleotide sequences of the 16S rRNA gene. Thermotolerant and halotolerant spore-forming bacteria 7t1 and 7t3 of terrestrial ecosystems Dead Sea identified as Bacillus licheniformis and B. subtilis subsp. subtilis, respectively. Taking into account remote location of thermotolerant strain 6t1 from closely related strains in the cluster Staphylococcus, 6t1 strain can be regarded as Staphylococcus sp. In terrestrial ecosystems, Galindez Island (Antarctic) detected taxonomically diverse psychrotolerant bacteria. From ornithogenic soil were isolated Micrococcus luteus O-1 and Microbacterium trichothecenolyticum O-3. Strains 4r5, 5r5 and 40r5, isolated from grass and lichens, can be referred to the genus Frondihabitans. These strains are taxonomically and ecologically isolated and on the tree diagram form the joint cluster with three isolates Frondihabitans sp., isolated from the lichen Austrian Alps, and psychrotolerant associated with plants F. cladoniiphilus CafT13(T). Isolates from black lichen in the different stationary observation points on the south side of a vertical cliff identified as: Rhodococcus fascians 181n3, Sporosarcina aquimarina O-7, Staphylococcus sp. 0-10. From orange biofilm of fouling on top of the vertical cliff isolated Arthrobacter sp. 28r5g1, from the moss-- Serratia sp. 6r1g. According to the results, Frondihabitans strains most frequently encountered among chemoorganotrophic aerobic bacteria in the Antarctic phytocenoses.

In this work we describe the conditional toxic effect of the expression of enzymes that cleave 5-bromo-4-chloro-3-indolyl (BCI) substrates and its use as a new counterselection principle useful for the generation of clean and unmarked mutations in the genomes of bacteria. The application of this principle was demonstrated in the thermophile Thermus thermophilus HB27 and in a mesophile for which currently no counterselection markers are available, Micrococcus luteus ATCC 27141. For T. thermophilus, the indigogenic substrate BCI-β-glucoside was used in combination with the T. thermophilus β-glucosidase gene (bgl). For M. luteus, a combination of BCI-β-galactoside and the E. coli lacZ gene was implemented. We observed a strong growth-inhibiting effect when the strains were grown on agar plates containing the appropriate BCI substrates, the inhibition being proportional to the substrate concentration and the level of bgl/lacZ expression. The growth inhibition apparently depends on intracellular BCI substrate cleavage and accumulation of toxic indoxyl precipitates. The bgl and lacZ genes were used as counterselection markers for the rapid generation of scar-less chromosomal deletions in T. thermophilus HB27 (both in a Δbgl and in a wild type background) and in M. luteus ATCC 27141. In addition to Thermus and Micrococcus, sensitivity to BCI substrate cleavage was observed for other Gram-negative and Gram-positive species belonging to various bacterial phyla, including representatives of the genera Staphylococcus, Bacillus, Corynebacterium, Rhodococcus, Paracoccus and Xanthomonas. Thus, the toxicity of indoxyl derivative accumulation upon BCI substrate cleavage can be used for selection purposes in a broad range of microorganisms., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

The momentum gained by research on biologics has not been met yet with equal thrust on the informatics side. There is a noticeable lack of software for data management that empowers the bench scientists working on the development of biologic therapeutics. SARvision|Biologics is a tool to analyze data associated with biopolymers, including peptides, antibodies, and protein therapeutics programs. The program brings under a single user interface tools to filter, mine, and visualize data as well as those algorithms needed to organize sequences. As part of the data-analysis tools, we introduce two new concepts: mutation cliffs and invariant maps. Invariant maps show the variability of properties when a monomer is maintained constant in a position of the biopolymer. Mutation cliff maps draw attention to pairs of sequences where a single or limited number of point mutations elicit a large change in a property of interest. We illustrate the program and its applications using a peptide data set collected from the literature.

Ralstonia eutropha is a facultatively chemolithoautotrophic bacterium able to grow with organic substrates or H2 and CO2 under aerobic conditions. Under conditions of nutrient imbalance, R. eutropha produces copious amounts of poly[(R)-3-hydroxybutyrate] (PHB). Its ability to utilize CO2 as a sole carbon source renders it an interesting new candidate host for the production of renewable liquid transportation fuels. We engineered R. eutropha for the production of fatty acid-derived, diesel-range methyl ketones. Modifications engineered in R. eutropha included overexpression of a cytoplasmic version of the TesA thioesterase, which led to a substantial (>150-fold) increase in fatty acid titer under certain conditions. In addition, deletion of two putative β-oxidation operons and heterologous expression of three genes (the acyl coenzyme A oxidase gene from Micrococcus luteus and fadB and fadM from Escherichia coli) led to the production of 50 to 65 mg/liter of diesel-range methyl ketones under heterotrophic growth conditions and 50 to 180 mg/liter under chemolithoautotrophic growth conditions (with CO2 and H2 as the sole carbon source and electron donor, respectively). Induction of the methyl ketone pathway diverted substantial carbon flux away from PHB biosynthesis and appeared to enhance carbon flux through the pathway for biosynthesis of fatty acids, which are the precursors of methyl ketones.

Functional variation of Rpf, a growth factor found exclusively in Actinobacteria, is differentiated by its source and amino acid sequences. Only purified Rpf proteins from three species have been studied so far. To seek new Rpfs for use in future studies to understand their role in Actinobacteria, the objective of this study was to identify rpf gene homologs in Tomitella biformata AHU 1821(T), a novel Actinobacteria isolated from permafrost ice wedge. Amplification using degenerate primers targeting the essential Rpf domain led to the discovery of a new rpf gene in T. biformata. Gene structure and the deduced Rpf domain amino acid sequence indicated that this rpf gene was not identical to previously studied Rpf. Phylogenetic analysis placed T. biformata Rpf in a monophyletic branch in the RpfB subfamily. The deduced amino acid sequence was 44.9% identical to RpfB in Mycobacterium tuberculosis, the closest functionally tested Rpf. The gene was cloned and expressed in Escherichia coli; the recombinant Rpf protein (rRpf) promoted the growth of dividing cells and resuscitated non-dividing cells of T. biformata. Compared to other studies, this Rpf was required at higher concentrations to promote its growth and to resuscitate itself from a non-dividing state. The resuscitation function was likely due to the highly conserved Rpf domain. This study provides evidence that a genetically unique but functional Rpf can be found in novel members of Actinobacteria and can lead to a better understanding of bacterial cytokines in this phylum.

The effective number of codons (N(c)) is a widely used index for characterizing codon usage bias because it does not require a set of reference genes as does codon adaptation index (CAI) and because of the freely available computational tools such as CodonW. However, N(c), as originally formulated has many problems. For example, it can have values far greater than the number of sense codons; it treats a 6-fold compound codon family as a single-codon family although it is made of a 2-fold and a 4-fold codon family that can be under dramatically different selection for codon usage bias; the existing implementations do not handle all different genetic codes; it is often biased by codon families with a small number of codons. We developed a new N(c) that has a number of advantages over the original N(c). Its maximum value equals the number of sense codons when all synonymous codons are used equally, and its minimum value equals the number of codon families when exactly one codon is used in each synonymous codon family. It handles all known genetic codes. It breaks the compound codon families (e.g., those involving amino acids coded by six synonymous codons) into 2-fold and 4-fold codon families. It reduces the effect of codon families with few codons by introducing pseudocount and weighted averages. The new N(c) has significantly improved correlation with CAI than the original N(c) from CodonW based on protein-coding genes from Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, Escherichia coli, Bacillus subtilis, Micrococcus luteus, and Mycoplasma genitalium. It also correlates better with protein abundance data from the yeast than the original N(c).

Novel vaccines are needed to control tuberculosis (TB), the bacterial infectious disease that together with malaria and HIV is worldwide responsible for high levels of morbidity and mortality. TB can result from the reactivation of an initially controlled latent infection by Mycobacterium tuberculosis (Mtb). Mtb proteins for which a possible role in this reactivation process has been hypothesized are the five homologs of the resuscitation-promoting factor of Micrococcus luteus, namely Mtb Rv0867c (rpfA), Rv1009 (rpfB), Rv1884c (rpfC), Rv2389c (rpfD) and Rv2450c (rpfE). Analysis of the immune recognition of these 5 proteins following Mtb infection or Mycobacterium bovis BCG vaccination of mice showed that Rv1009 (rpfB) and Rv2389c (rpfD) are the most antigenic in the tested models. We therefore selected rpfB and rpfD for testing their vaccine potential as plasmid DNA vaccines. Elevated cellular immune responses and modest but significant protection against intra-tracheal Mtb challenge were induced by immunization with the rpfB encoding DNA vaccine. The results indicate that rpfB is the most promising candidate of the five rpf-like proteins of Mtb in terms of its immunogenicity and protective efficacy and warrants further analysis for inclusion as an antigen in novel TB vaccines., (Copyright © 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Objective: The purpose of the present study was to produce the Rpf (resuscitation promoting factor) protein by cloning and expressing the rpf gene, secreted by Micrococcus luteus IAM 14879, in Escherichia coli and to evaluate its role in the recovery of the VBNC (viable but non-culturable) state in high-GC Gram-positive bacteria., Methods: Genomic DNA was extracted from Micrococcus luteus IAM 14879 and the rpf gene was amplified by PCR using specific primers. The PCR products was purified, cloned into a pET15b expression vector, and transformed into Escherichia coli BL21 (DE3). Then the pET15b plasmid expression vector was used to confirm the purification of the recombinant proteins via SDS-PAGE. The VBNC state cells from the high-GC Gram-positive bacteria, Rhodococcus sp. DS471, were used to confirm the promotion and recovery of growth capacity. Rhodococcus sp. DS471 were isolated from soil and closely related to Micrococcus luteus IAM 14879., Results: The gene sequences confirmed that the rpf gene from Micrococcus luteus IAM 14879 that was expressed in Escherichia coli, was 672 bp. SDS-PAGE analysis showed that the recombinant Rpf protein was obtained successfully, and further studies showed it capable of promoting the recovery of the VBNC state by about 100-fold relative to the control., Conclusion: Rpf of Micrococus luteus IAM 14879 can be successfully cloned and expressed in Escherichia coli and shows a strong ability to promote the recovery of the VBNC state of cells of Rhodococcus sp. DS471.

The inducible Pm promoter integrated into broad-host-range plasmid RK2 replicons can be fine-tuned continuously between the uninduced and maximally induced levels by varying the inducer concentrations. To lower the uninduced background level while still maintaining the inducibility for applications in, for example, metabolic engineering and synthetic (systems) biology, we report here the use of mutations in the Pm DNA region corresponding to the 5' untranslated region of mRNA (UTR). Five UTR variants obtained by doped oligonucleotide mutagenesis and selection, apparently reducing the efficiency of translation, were all found to display strongly reduced uninduced expression of three different reporter genes (encoding β-lactamase, luciferase, and phosphoglucomutase) in Escherichia coli. The ratio between induced and uninduced expression remained the same or higher compared to cells containing a corresponding plasmid with the wild-type UTR. Interestingly, the UTR variants also displayed similar effects on expression when substituted for the native UTR in another and constitutive promoter, P1 (P(antitet)), indicating a broad application potential of these UTR variants. Two of the selected variants were used to control the production of the C(50) carotenoid sarcinaxanthin in an engineered strain of E. coli that produces the precursor lycopene. Sarcinaxanthin is produced in this particular strain by expressing three Micrococcus luteus derived genes from the promoter Pm. The results indicated that UTR variants can be used to eliminate sarcinaxanthin production under uninduced conditions, whereas cells containing the corresponding plasmid with a wild-type UTR produced ca. 25% of the level observed under induced conditions.

Different strains of Micrococcus luteus, isolated from high-altitude Argentinean wetlands, were recently reported to harbour the linear plasmids pLMA1, pLMH5 and pLMV7, all of which with 5'-covalently attached terminal proteins. The link between pLMA1 and the host's erythromycin resistance as well as further presumptive qualities prompted us to perform a detailed characterization. When the 454 technology was applied for direct sequencing of gel-purified pLMA1, assembly of the reads was impossible. However, combined Sanger/454 sequencing of cloned pLMA1 fragments, covering altogether 23 kb of the 110-kb spanning plasmid, allowed numerous sequence repeats of varying in lengths to be identified thus rendering an explanation for the above 454 assembly failure. A large number of putative transposase genes were identified as well. Furthermore, a region with five putative iteron sequences is possibly involved in pLMA1 replication.

We report the cloning and characterization of the biosynthetic gene cluster (crtE, crtB, crtI, crtE2, crtYg, crtYh, and crtX) of the γ-cyclic C(50) carotenoid sarcinaxanthin in Micrococcus luteus NCTC2665. Expression of the complete and partial gene cluster in Escherichia coli hosts revealed that sarcinaxanthin biosynthesis from the precursor molecule farnesyl pyrophosphate (FPP) proceeds via C(40) lycopene, C(45) nonaflavuxanthin, C(50) flavuxanthin, and C(50) sarcinaxanthin. Glucosylation of sarcinaxanthin was accomplished by the crtX gene product. This is the first report describing the biosynthetic pathway of a γ-cyclic C(50) carotenoid. Expression of the corresponding genes from the marine M. luteus isolate Otnes7 in a lycopene-producing E. coli host resulted in the production of up to 2.5 mg/g cell dry weight sarcinaxanthin in shake flasks. In an attempt to experimentally understand the specific difference between the biosynthetic pathways of sarcinaxanthin and the structurally related ε-cyclic decaprenoxanthin, we constructed a hybrid gene cluster with the γ-cyclic C(50) carotenoid cyclase genes crtYg and crtYh from M. luteus replaced with the analogous ε-cyclic C(50) carotenoid cyclase genes crtYe and crtYf from the natural decaprenoxanthin producer Corynebacterium glutamicum. Surprisingly, expression of this hybrid gene cluster in an E. coli host resulted in accumulation of not only decaprenoxanthin, but also sarcinaxanthin and the asymmetric ε- and γ-cyclic C(50) carotenoid sarprenoxanthin, described for the first time in this work. Together, these data contributed to new insight into the diverse and multiple functions of bacterial C(50) carotenoid cyclases as key catalysts for the synthesis of structurally different carotenoids.

Balloon flower (Platycodon grandiflorum) is widely cultivated vegetable and used as a remedy for asthma in East Asia. Experiments were conducted to isolate endophytic bacteria from 1-, 3-, and 6-year-old balloon flower roots and to analyze the enzymatic, antifungal, and anti-human pathogenic activities of the potential endophytic biocontrol agents obtained. Total 120 bacterial colonies were isolated from the interior of all balloon flower roots samples. Phylogenetic analysis based on 16S rRNA gene sequences showed that the population of 'low G + C gram-positive bacteria' (LGCGPB) gradually increased 60.0-80.0% from 1 to 6 years balloon flower sample. On the other hand, maximum hydrolytic enzyme activity showing endophytic bacteria was under LGCGPB, among the bacterial strains, Bacillus sp. (BF1-1 and BF3-8), Bacillus sp. (BF1-2 and BF3-5), and Bacillus sp. (BF1-3, BF3-6, and BF6-4) showed maximum enzyme activities. Besides, Bacillus licheniformis (BF3-5 and BF6-6) and Bacillus pumilus (BF6-1) showed maximum antifungal activity against Phytophthora capsici, Fusarium oxysporum, Rhizoctonia solani, and Pythium ultimum. Moreover, Bacillus licheniformis was found in 3 and 6 years balloon flower roots, but Bacillus pumilus was found only in 6 years sample. It is presumed that older balloon flower plants invite more potential antifungal endophytes for there protection from plant diseases. In addition, Bacillus sp. (BF1-2 and BF3-5) showed maximum anti-human pathogenic activity. So, plant age is presumed to influence diversity of balloon flower endophytic bacteria.

The aim of this study was to demonstrate the presence of yeast and bacterial biofilms on the surface of tracheoesophageal voice prostheses (TVPs) by a double-staining technique with confocal laser scanning microscopy (CLSM). Biofilms of 12 removed TVPs were visualized by scanning electron microscopy, then stained with ConA-FITC and propidium iodide for CLSM. Microbial identification was by partial 16S rRNA gene analysis and ITS-2 sequence analysis. Microbial biofilms on the TVPs consisted of bacteria and filamentous cells. Bacterial cells were attached to the filamentous and unicellular yeast cells, thus forming a network. Sequence analyses of six voice prostheses identified the presence of a variety of bacterial and yeast species. In vivo studies showed that Klebsiella oxytoca and Micrococcus luteus efficiently attached to Candida albicans. CLSM with double fluorescence staining can be used to demonstrate biofilm formations composed of a mixture of yeast and bacterial cells on the surface of TVPs.

The carcinogenic action of UVA radiation is commonly attributed to DNA oxidation mediated by endogenous photosensitisers. Yet, it was recently shown that cyclobutane pyrimidine dimers (CPD), well known for their involvement in UVB genotoxicity, are produced in larger yield than oxidative lesions in UVA-irradiated cells and skin. In the present work, we gathered mechanistic information on this photoreaction by comparing formation of all possible bipyrimidine photoproducts upon UVA irradiation of cells, purified genomic DNA and dA(20):dT(20) oligonucleotide duplex. We observed that the distribution of photoproducts, characterized by the sole formation of CPD and the absence of (6-4) photoproducts was similar in the three types of samples. The CPD involving two thymines represented 90% of the amount of photoproducts. Moreover, the yields of formation of the DNA lesions were similar in cells and isolated DNA. In addition, the effect of the wavelength of the incident photons was found to be the same in isolated DNA and cells. This set of data shows that UVA-induced cyclobutane pyrimidine dimers are formed via a direct photochemical mechanism, without mediation of a cellular photosensitiser. This is possible because the double-stranded structure increases the capacity of DNA bases to absorb UVA photons, as evidenced in the case of the oligomer dA(20):dT(20). These results emphasize the need to consider UVA in the carcinogenic effects of sunlight. An efficient photoprotection is needed that can only be complete by completely blocking incident photons, rather than by systemic approaches such as antioxidant supplementation.

Micrococcus luteus (NCTC2665, "Fleming strain") has one of the smallest genomes of free-living actinobacteria sequenced to date, comprising a single circular chromosome of 2,501,097 bp (G+C content, 73%) predicted to encode 2,403 proteins. The genome shows extensive synteny with that of the closely related organism, Kocuria rhizophila, from which it was taxonomically separated relatively recently. Despite its small size, the genome harbors 73 insertion sequence (IS) elements, almost all of which are closely related to elements found in other actinobacteria. An IS element is inserted into the rrs gene of one of only two rrn operons found in M. luteus. The genome encodes only four sigma factors and 14 response regulators, a finding indicative of adaptation to a rather strict ecological niche (mammalian skin). The high sensitivity of M. luteus to beta-lactam antibiotics may result from the presence of a reduced set of penicillin-binding proteins and the absence of a wblC gene, which plays an important role in the antibiotic resistance in other actinobacteria. Consistent with the restricted range of compounds it can use as a sole source of carbon for energy and growth, M. luteus has a minimal complement of genes concerned with carbohydrate transport and metabolism and its inability to utilize glucose as a sole carbon source may be due to the apparent absence of a gene encoding glucokinase. Uniquely among characterized bacteria, M. luteus appears to be able to metabolize glycogen only via trehalose and to make trehalose only via glycogen. It has very few genes associated with secondary metabolism. In contrast to most other actinobacteria, M. luteus encodes only one resuscitation-promoting factor (Rpf) required for emergence from dormancy, and its complement of other dormancy-related proteins is also much reduced. M. luteus is capable of long-chain alkene biosynthesis, which is of interest for advanced biofuel production; a three-gene cluster essential for this metabolism has been identified in the genome.

Dormancy among nonsporulating actinobacteria is now a widely accepted phenomenon. In Micrococcus luteus, the resuscitation of dormant cells is caused by a small secreted protein (resuscitation-promoting factor, or Rpf) that is found in "spent culture medium." Rpf is encoded by a single essential gene in M. luteus. Homologs of Rpf are widespread among the high G + C Gram-positive bacteria, including mycobacteria and streptomycetes, and most organisms make several functionally redundant proteins. M. luteus Rpf comprises a lysozyme-like domain that is necessary and sufficient for activity connected through a short linker region to a LysM motif, which is present in a number of cell-wall-associated enzymes. Muralytic activity is responsible for resuscitation. In this report, we characterized a number of environmental isolates of M. luteus, including several recovered from amber. There was substantial variation in the predicted rpf gene product. While the lysozyme-like and LysM domains showed little variation, the linker region was elongated from ten amino acid residues in the laboratory strains to as many as 120 residues in one isolate. The genes encoding these Rpf proteins have been characterized, and a possible role for the Rpf linker in environmental adaptation is proposed. The environmental isolates show enhanced resistance to lysozyme as compared with the laboratory strains and this correlates with increased peptidoglycan acetylation. In strains that make a protein with an elongated linker, Rpf was bound to the cell wall, rather than being released to the growth medium, as occurs in reference strains. This rpf gene was introduced into a lysozyme-sensitive reference strain. Both rpf genes were expressed in transformants which showed a slight but statistically significant increase in lysozyme resistance.

Aliphatic hydrocarbons are highly appealing targets for advanced cellulosic biofuels, as they are already predominant components of petroleum-based gasoline and diesel fuels. We have studied alkene biosynthesis in Micrococcus luteus ATCC 4698, a close relative of Sarcina lutea (now Kocuria rhizophila), which 4 decades ago was reported to biosynthesize iso- and anteiso-branched, long-chain alkenes. The underlying biochemistry and genetics of alkene biosynthesis were not elucidated in those studies. We show here that heterologous expression of a three-gene cluster from M. luteus (Mlut&#95;13230-13250) in a fatty acid-overproducing Escherichia coli strain resulted in production of long-chain alkenes, predominantly 27:3 and 29:3 (no. carbon atoms: no. C=C bonds). Heterologous expression of Mlut&#95;13230 (oleA) alone produced no long-chain alkenes but unsaturated aliphatic monoketones, predominantly 27:2, and in vitro studies with the purified Mlut&#95;13230 protein and tetradecanoyl-coenzyme A (CoA) produced the same C(27) monoketone. Gas chromatography-time of flight mass spectrometry confirmed the elemental composition of all detected long-chain alkenes and monoketones (putative intermediates of alkene biosynthesis). Negative controls demonstrated that the M. luteus genes were responsible for production of these metabolites. Studies with wild-type M. luteus showed that the transcript copy number of Mlut&#95;13230-13250 and the concentrations of 29:1 alkene isomers (the dominant alkenes produced by this strain) generally corresponded with bacterial population over time. We propose a metabolic pathway for alkene biosynthesis starting with acyl-CoA (or-ACP [acyl carrier protein]) thioesters and involving decarboxylative Claisen condensation as a key step, which we believe is catalyzed by OleA. Such activity is consistent with our data and with the homology (including the conserved Cys-His-Asn catalytic triad) of Mlut&#95;13230 (OleA) to FabH (beta-ketoacyl-ACP synthase III), which catalyzes decarboxylative Claisen condensation during fatty acid biosynthesis.

During the period 2002-2008, at the National Cardiovascular Center, Osaka, 28 Micrococcus luteus isolates and one Kocuria spp. isolate were obtained from blood cultures of pulmonary hypertension (PH) patients who were receiving continuous infusion therapy with epoprostenol. Pulsed-field gel electrophoresis patterns of the isolates were unrelated, suggesting that the infections had multiple origins. The preparation of epoprostenol solution by patients themselves was thought to be a risk factor.

Iron is indispensable to the growth and metabolism of all marine organisms, including bacteria. In this work, we investigated and compared the influence of iron(III) concentration on the growth of and siderophore production by two heterotrophic bacteria--Micrococcus luteus and Bacillus silvestris. Our results showed that the iron concentration strongly influences the growth of both species. The growth curves were different for each iron concentration and each strain. M. luteus grew more rapidly than B. silvestris, but produced a roughly four times smaller quantity of siderophores. Both M. luteus and B. silvestris secreted hydroxamate-type siderophores and alpha-keto/alpha-hydroxy acids, but did not produce catecholates. This paper is probably the first to report on siderophore production by B. silvestris and M. luteus isolated from seawater. Moreover, the influence of different iron concentrations on the growth of and siderophore production in these bacteria has been documented. This provides further evidence indicating iron bioavailability as the actual reason for siderophore release by biota.

cis-Prenyltransferases catalyze the consecutive condensation of isopentenyl diphosphate (IPP) with allylic prenyl diphosphates, producing Z,E-mixed prenyl diphosphate. The Mycobacterium tuberculosis Z,E-farnesyl diphosphate synthase Rv1086 catalyzes the condensation of one molecule of IPP with geranyl diphosphate to yield Z,E-farnesyl diphosphate and is classified as a short-chain cis-prenyltransferase. To elucidate the chain-length determination mechanism of the short-chain cis-prenyltransferase, we introduced some substitutive mutations at the characteristic amino acid residues of Rv1086. Among the mutants constructed, L84A showed a dramatic change of catalytic function to synthesize longer prenyl chain products than that of wild type, indicating that Leu84 of Rv1086 plays an important role in product chain-length determination. Mutagenesis at the corresponding residue of a medium-chain cis-prenyltransferase, Micrococcus luteus B-P 26 undecaprenyl diphosphate synthase also resulted in the production of different prenyl chain length from the intrinsic product, suggesting that this position also plays an important role in product chain-length determination for medium-chain cis-prenyltransferases.

Gordonia westfalica Kb1 and Gordonia polyisoprenivorans VH2 induce the formation of an extracellular superoxide dismutase (SOD) during poly(cis-1,4-isoprene) degradation. To investigate the function of this enzyme in G. polyisoprenivorans VH2, the sodA gene was disrupted. The mutants exhibited reduced growth in liquid mineral salt media containing poly(cis-1,4-isoprene) as the sole carbon and energy source, and no SOD activity was detectable in the supernatants of the cultures. Growth experiments revealed that SodA activity is required for optimal growth on poly(cis-1,4-isoprene), whereas this enzyme has no effect on aerobic growth in the presence of water-soluble substrates like succinate, acetate, and propionate. This was detected by activity staining, and proof of expression was by antibody detection of SOD. When SodA from G. westfalica Kb1 was heterologously expressed in the sodA sodB double mutant Escherichia coli QC779, the recombinant mutant exhibited increased resistance to paraquat, thereby indicating the functionality of the G. westfalica Kb1 SodA and indirectly protection of G. westfalica cells by SodA from oxidative damage. Both sodA from G. polyisoprenivorans VH2 and sodA from G. westfalica Kb1 coded for polypeptides comprising 209 amino acids and having approximately 90% and 70% identical amino acids, respectively, to the SodA from Mycobacterium smegmatis strain MC(2) 155 and Micrococcus luteus NCTC 2665. As revealed by activity staining experiments with the wild type and the disruption mutant of G. polyisoprenivorans, this bacterium harbors only one active SOD belonging to the manganese family. The N-terminal sequences of the extracellular SodA proteins of both Gordonia species showed no evidence of leader peptides for the mature proteins, like the intracellular SodA protein of G. polyisoprenivorans VH2, which was purified under native conditions from the cells. In G. westfalica Kb1 and G. polyisoprenivorans VH2, SodA probably provides protection against reactive oxygen intermediates which occur during degradation of poly(cis-1,4-isoprene).

Objective: To express Micrococcus luteus resuscitation promoting factor (Rpf) domain and its mutants in prokaryotic cells, and to investigate their bioactivity., Methods: The gene of Rpf domain and its mutants (E54K, E54A) were amplified by polymerase chain reaction (PCR) from the genome of Micrococcus luteus and cloned into pMD18-T vector. After sequenced, the Rpf domain and its mutant gene were subcloned into expression vector PGEX-4T-1, and transfected into E. coli DH5alpha. The expressed product was purified by affinity chromatography using GST Fusion Protein Purification bead. The aim proteins were identified by SDS-PAGE analysis and by Western blot with monoclonal antibodies against Rpf domain (mAb). The bioactivity of the proteins was analyzed by stimulating the resuscitation of Mycobacterium smegmatis., Results: The sequences of the PCR products were identical to those of the Rpf domain and its mutant gene in GenBank. The relative molecular mass identified by SDS-PAGE analysis was consistent with that had been reported, which was also confirmed by Western blot analysis that there were specific bindings at 32 000 with Rpf domain mAb. The purified GST-Rpf domain could stimulate resuscitation of Mycobacterium smegmatis. Replacements E54A and especially E54K resulted in inhibition of Rpf resuscitation activity., Conclusions: Rpf domain and two kinds of its mutant protein were obtained, and its effects on the resuscitation of dormant Mycobacterium smegmatis were clarified.

Benzylisoquinoline alkaloids, such as the analgesic compounds morphine and codeine, and the antibacterial agents berberine, palmatine, and magnoflorine, are synthesized from tyrosine in the Papaveraceae, Berberidaceae, Ranunculaceae, Magnoliaceae, and many other plant families. It is difficult to produce alkaloids on a large scale under the strict control of secondary metabolism in plants, and they are too complex for cost-effective chemical synthesis. By using a system that combines microbial and plant enzymes to produce desired benzylisoquinoline alkaloids, we synthesized (S)-reticuline, the key intermediate in benzylisoquinoline alkaloid biosynthesis, from dopamine by crude enzymes from transgenic Escherichia coli. The final yield of (S)-reticuline was 55 mg/liter within 1 h. Furthermore, we synthesized an aporphine alkaloid, magnoflorine, or a protoberberine alkaloid, scoulerine, from dopamine via reticuline by using different combination cultures of transgenic E. coli and Saccharomyces cerevisiae cells. The final yields of magnoflorine and scoulerine were 7.2 and 8.3 mg/liter culture medium. These results indicate that microbial systems that incorporate plant genes cannot only enable the mass production of scarce benzylisoquinoline alkaloids but may also open up pathways for the production of novel benzylisoquinoline alkaloids.

Genetic markers have been in popular use for tracing microbial movement in the environment. However, the impact of genetic marker insertion on microbial surface properties and consequent transport is often ignored. For this research, we investigated the impact of luminescence-based genetic marker insertion on bacterial surface properties and transport. Typical Gram-positive bacterial strains of Lactobacillus casei, Streptococcus mitis and Micrococcus luteus were used as model bacterial strains in this research. We manipulated gene transfer to observe the impact of lux gene insertion on bacterial surface properties based on contact angle measurements, and we conducted column experiments to evaluate the impact of lux gene insertion on bacterial transport. After lux gene insertion, bacterial interactions with the porous media increased, demonstrating stronger deposition potential in the porous media. Accordingly, retention of the daughter strains increased. Lux gene insertion also resulted in an increase in bacterial dispersion and equilibrium adsorption in the porous media. The bacterial deposition coefficient was found to correlate with the free energy of interactions between bacteria and the porous media.

DNA-based stable isotope probing (SIP) is a novel technique for the identification of organisms actively assimilating isotopically labeled compounds. Herein, we define the limitations to using 15N-labeled substrates for SIP and propose modifications to compensate for these shortcomings. Changes in DNA buoyant density (BD) resulting from 15N incorporation were determined using cultures of disparate GC content (Escherichia coli and Micrococcus luteus). Incorporation of 15N into DNA increased BD by 0.015+/-0.002 g mL(-1) for E. coli and 0.013+/-0.002 g mL(-1) for M. luteus. The DNA BD shift was greatly increased (0.045 g mL(-1)) when dual isotope (13C plus 15N) labeling was employed. Despite the limited DNA BD shift following 15N enrichment, we found the use of gradient fractionation, followed by a comparison of T-RFLP profiles from fractions of labeled and control treatments, facilitated detection of enrichment in DNA samples from either cultures or soil.

Aspergillus oryzae RIB40 possesses the gene of glutaminase (Micrococcus luteus K-3-type glutaminase; AoGls), which has 40% homology with the salt-tolerant glutaminase from M. luteus K-3 (Micrococcus glutaminase). It was found that AoGls is a salt-tolerant enzyme, and its properties are similar to those of Micrococcus glutaminase.