A polyesterase from the Antarctic bacterium Moraxella sp. degrades highly crystalline synthetic polymers.

The uncontrolled release of plastics in the environment has rendered them ubiquitous around the planet, threatening the wildlife and human health. Biodegradation and valorization of plastics has emerged as an eco-friendly alternative to conventional management techniques. Discovery of novel polymer-degrading enzymes with diversified properties is hence an important task in order to explore different operational conditions for plastic-waste upcycling. In the present study, a barely studied psychrophilic enzyme (MoPE) from the Antractic bacterium Moraxella sp. was heterologously expressed, characterized and its potential in polymer degradation was further investigated. Based on its amino acid composition and structure, MoPE resembled PET-degrading enzymes, sharing features from both mesophilic and thermophilic homologues. MoPE hydrolyzes non-biodegradable plastics, such as polyethylene terephthalate and polyurethane, as well as biodegradable synthetic polyesters, such as polycaprolactone, polyhydroxy butyrate, polybutylene succinate and polylactic acid. The mass fraction crystallinity of the aliphatic polymers tested ranged from 11% to 64% highlighting the potential of the enzyme to hydrolyze highly crystalline plastics. MoPE was able to degrade different types of amorphous and semi-crystalline PET, releasing water-soluble monomers and showed synergy with a feruloyl esterase of the tannase family for the release of terephthalic acid. Based on the above, MoPE was characterized as a versatile psychrophilic polyesterase demonstrating a broad-range plastics degradation potential.

Interspecies Outer Membrane Vesicles (OMVs) Modulate the Sensitivity of Pathogenic Bacteria and Pathogenic Yeasts to Cationic Peptides and Serum Complement.

The virulence of bacterial outer membrane vesicles (OMVs) contributes to innate microbial defense. Limited data report their role in interspecies reactions. There are no data about the relevance of OMVs in bacterial-yeast communication. We hypothesized that model Moraxella catarrhalis OMVs may orchestrate the susceptibility of pathogenic bacteria and yeasts to cationic peptides (polymyxin B) and serum complement. Using growth kinetic curve and time-kill assay we found that OMVs protect Candida albicans against polymyxin B-dependent fungicidal action in combination with fluconazole. We showed that OMVs preserve the virulent filamentous phenotype of yeasts in the presence of both antifungal drugs. We demonstrated that bacteria including Haemophilus influenza, Acinetobacter baumannii, and Pseudomonas aeruginosa coincubated with OMVs are protected against membrane targeting agents. The high susceptibility of OMV-associated bacteria to polymyxin B excluded the direct way of protection, suggesting rather the fusion mechanisms. High-performance liquid chromatography-ultraviolet spectroscopy (HPLC-UV) and zeta-potential measurement revealed a high sequestration capacity (up to 95%) of OMVs against model cationic peptide accompanied by an increase in surface electrical charge. We presented the first experimental evidence that bacterial OMVs by sequestering of cationic peptides may protect pathogenic yeast against combined action of antifungal drugs. Our findings identify OMVs as important inter-kingdom players.

Analysis of microbiome in raw chicken meat from butcher shops and packaged products in South Korea to detect the potential risk of foodborne illness.

Chicken meat is one of the most widely consumed meats worldwide. The microbiota on the whole body of chicken is a potential source of foodborne pathogens that can be transmitted to humans during the preparation of raw meat. However, to date, there have been no studies comparing the microbiota of packaged chicken products and those of raw chicken carcasses from butcher shops, although such information could be useful for identifying sources of contamination in cases of food poisoning. We addressed this in the present study by analyzing the microbiota of 80 chicken meat samples collected from various butcher shops and processing plants in South Korea with the Illumina MiSeq system based on the 16S rRNA gene sequence. The bacterial amounts in chicken samples were estimated by quantitative real-time PCR. Although different microbial members were present in unpackaged meat from butcher shops as compared to those in packaged products from commercial sources, seasonal differences (sample obtained in January vs. July) in microbiota were more significant even in the packaged products from the same company. We also investigated the influence of contaminated foodborne pathogen on the indigenous microbiota (64 chicken samples) by artificially inoculated with Salmonella enterica serotype Virchow on chicken carcasses under various conditions, and carrying out 16S rRNA gene and whole metagenome sequencing. The amount of contaminated Salmonella in chicken meat samples was the highest and lowest in samples stored at 27 °C and 4 °C after washing, respectively. Additionally, the relative abundance of virulence genes was detected lower in samples stored at 4 °C after washing in both butcher shop and commercial samples. These results could be useful for reducing the risk of foodborne illness caused by cross-contamination during the preparation of chicken meat.

An anti-CRISPR protein disables type V Cas12a by acetylation.

Phages use anti-CRISPR proteins to deactivate the CRISPR-Cas system. The mechanisms for the inhibition of type I and type II systems by anti-CRISPRs have been elucidated. However, it has remained unknown how the type V CRISPR-Cas12a (Cpf1) system is inhibited by anti-CRISPRs. Here we identify the anti-CRISPR protein AcrVA5 and report the mechanisms by which it inhibits CRISPR-Cas12a. Our structural and biochemical data show that AcrVA5 functions as an acetyltransferase to modify Moraxella bovoculi (Mb) Cas12a at Lys635, a residue that is required for recognition of the protospacer-adjacent motif. The AcrVA5-mediated modification of MbCas12a results in complete loss of double-stranded DNA (dsDNA)-cleavage activity. In contrast, the Lys635Arg mutation renders MbCas12a completely insensitive to inhibition by AcrVA5. A cryo-EM structure of the AcrVA5-acetylated MbCas12a reveals that Lys635 acetylation provides sufficient steric hindrance to prevent dsDNA substrates from binding to the Cas protein. Our study reveals an unprecedented mechanism of CRISPR-Cas inhibition and suggests an evolutionary arms race between phages and bacteria.

Biodegradation of Tributyltin (TBT) by Extremophile Bacteria from Atacama Desert and Speciation of Tin By-products.

Biodegradation of tributyltin (TBT) by four tin resistant Gram negative bacteria isolated from extremely contaminated river sediments in the Atacama Desert in Chile was studied. Moraxella osloensis showed the greatest resistance and degradation capability of TBT, producing less toxic by-products, such as dibutyltin (DBT) and inorganic tin. In 7 days, approximately 80 % of TBT degradation was achieved, generating close to 20 % of DBT as degradation product. The degradation rate constant (k) was 0.022 [day(-1)] and TBT half-life (t1/2) in culture was 4.3 days. Debutylation is stated a probable mechanism of TBT degradation.

Biodegradation of the textile dye Mordant Black 17 (Calcon) by Moraxella osloensis isolated from textile effluent-contaminated site.

The bacterium with dye degrading ability was isolated from effluent disposal sites of textile industries, Tirupur and was identified as Moraxella osloensis based on the biochemical and morphological characterization as well as 16S rRNA sequencing. This organism was found to decolorize 87 % of Mordant Black 17 at 100 mg l⁻¹ under shake culture condition compared to 92 % under stationary culture condition. Maximum degradation of the dye by M. osloensis was achieved when the mineral salt medium was supplemented with 0.5 % glucose and 0.1 % ammonium nitrate at 35 °C. Degradation of dye was found to follow first order kinetics with the k value of 0.06282 h⁻¹ and a R² value of 0.955. Analyses for the identification of intermediate compounds confirmed the presence of naphthalene, naphthol, naphthoquinone, salicylic acid and catechol. Based on this finding a probable pathway for the degradation of Mordant Black 17 by M. osloensis has been proposed.

Fermentation products of solvent tolerant marine bacterium Moraxella spp. MB1 and its biotechnological applications in salicylic acid bioconversion.

As part of a proactive approach to environmental protection, emerging issues with potential impact on the environment is the subject of ongoing investigation. One emerging area of environmental research concerns pharmaceuticals like salicylic acid, which is the main metabolite of various analgesics including aspirin. It is a common component of sewage effluent and also an intermediate in the degradation pathway of various aromatic compounds which are introduced in the marine environment as pollutants. In this study, biotransformation products of salicylic acid by seaweed, Bryopsis plumosa, associated marine bacterium, Moraxella spp. MB1, have been investigated. Phenol, conjugates of phenol and hydroxy cinnamic acid derivatives (coumaroyl, caffeoyl, feruloyl and trihydroxy cinnamyl) with salicylic acid (3-8) were identified as the bioconversion products by electrospray ionization mass spectrometry. These results show that the microorganism do not degrade phenolic acid but catalyses oxygen dependent transformations without ring cleavage. The degradation of salicylic acid is known to proceed either via gentisic acid pathway or catechol pathway but this is the first report of biotransformation of salicylic acid into cinnamates, without ring cleavage. Besides cinnamic acid derivatives (9-12), metabolites produced by the bacterium include antimicrobial indole (13) and ß-carbolines, norharman (14), harman (15) and methyl derivative (16), which are beneficial to the host and the environment.

Moraxella species are primarily responsible for generating malodor in laundry.

Many people in Japan often detect an unpleasant odor generated from laundry that is hung to dry indoors or when using their already-dried laundry. Such an odor is often described as a "wet-and-dirty-dustcloth-like malodor" or an "acidic or sweaty odor." In this study, we isolated the major microorganisms associated with such a malodor, the major component of which has been identified as 4-methyl-3-hexenoic acid (4M3H). The isolates were identified as Moraxella osloensis by morphological observation and biochemical and phylogenetic tree analyses. M. osloensis has the potential to generate 4M3H in laundry. The bacterium is known to cause opportunistic infections but has never been known to generate a malodor in clothes. We found that M. osloensis exists at a high frequency in various living environments, particularly in laundry in Japan. The bacterium showed a high tolerance to desiccation and UV light irradiation, providing one of the possible reasons why they survive in laundry during and even after drying.

Novel detection of Escherichia coli beta-D-glucuronidase activity using a microbially-modified glassy carbon electrode and its potential for faecal pollution monitoring.

The electrochemical detection of Escherichia coli beta-D-glucuronidase activity as a means of monitoring water pollution by faecal material was investigated using separate Moraxella- and Pseudomonas putida-modified glassy carbon electrodes. The former was more sensitive and selective. The Moraxella-modified biosensor was 100 times more rapid and sensitive than the spectrophotometric detection of beta-D-glucuronidase activity. The experimental limit of detection of the biosensor was two c.f.u. per 100 ml polluted water sample within 20 min. The biosensor gave a linear response to commercial beta-D-glucuronidase concentration between 0.2 ng and 2 microg ml(-1). The biosensor detected activity of beta-D-glucuronidase from viable but non-culturable (VBNC) cells and can therefore serve as a presence or absence device for rapid water quality monitoring.

Biotransformation of citrinin to decarboxycitrinin using an organic solvent-tolerant marine bacterium, Moraxella sp. MB1.

Organic solvent tolerant microorganisms (OSTMs) are novel group of extremophilic microorganisms that have developed resistance to withstand solvent toxicity. These organisms play an important role in biotransformation of organic compounds. In the present study, we used an organic solvent-tolerant marine bacterium, Moraxella sp. MB1. 16S rRNA sequencing revealed that the bacterium shows 98% similarity with an uncultured marine bacterium with GenBank accession no. AY936933. This bacterium was used for the transformation of a toxin, citrinin, into decarboxycitrinin in a biphasic system. This transformation was affected by decarboxylase enzyme produced by MB1. Transformation of citrinin to decarboxycitrinin was monitored by thin-layer chromatography (TLC) and spectrophotometrically. Citrinin decarboxylase activity responsible for transformation was studied in cell-free growth medium and cell lysate of Moraxella sp. MB1. Citrinin decarboxylase was found to be intracellular in nature. The biotransformed product was purified and identified as decarboxycitrinin using electrospray ionization mass spectrometry (ESI-MS/MS) and nuclear magnetic resonance (NMR) spectrometry. The antibiotic activity of both citrinin and decarboxycitrinin is also reported.

Effect of carbon starvation on p-nitrophenol degradation by a Moraxella strain in buffer and river water.

This study examines the effect of carbon starvation on the ability of a Moraxella sp. strain to degrade p-nitrophenol (PNP). Carbon starvation for 24 h decreased the induction time for p-nitrophenol degradation by the bacterium in a minimal salt medium from 6 to 1 h but it did not completely eliminate the induction time. Moraxella cells with 2-day carbon starvation had an induction time of 3 h and the induction time of the 3-day starved cells was 6 h. A 100% increase in density of the non-starved cells did not affect the induction time for p-nitrophenol degradation by the bacterium, indicating that the initial increase in cell density of the carbon-starved culture did not cause the faster onset of p-nitrophenol degradation. However, the initial uptake of p-nitrophenol of the 1-day carbon-starved Moraxella cells was 3-fold higher than the non-starved cells. A green fluorescent protein gene (gfp)-labelled Moraxella (M6 strain) was constructed to examine the survival of and p-nitrophenol degradation by the bacterium in non-sterile river water samples. Similar p-nitrophenol degradation behaviour was observed in the river water samples inoculated with the M6 cells. The time needed for complete degradation of p-nitrophenol by the non-starved M6 was 19-27 and 33 h in samples spiked with 80, 200 and 360 microM p-nitrophenol, respectively. However, the 1-day carbon-starved inocula required about 16 h to degrade the p-nitrophenol completely regardless of its concentration in the water samples. Survival of the carbon-starved and non-starved M6 was not significantly different from each other in the river water regardless of the p-nitrophenol concentration. In the absence of p-nitrophenol, the inoculum density decreased continuously. At 200 and 360 microM p-nitrophenol, the cell densities of M6 increased in the first two days of incubation and declined steadily afterward.

Stress-survival responses of a carbon-starved p-nitrophenol-mineralizing Moraxella strain in river water.

The effect of carbon starvation on the stress-resistant responses of a p-nitrophenol-mineralizing Moraxella strain was examined in both buffer and river water samples. The Moraxella strain showed optimal stress-resistant responses in a minimal salt buffer when carbon-starved for 1-2 d. In the buffer system, the 1- and 2-day carbon-starved Moraxella cultures survived about 150-, 200-, and 100-fold better than the non-starved cultures when exposed to 43.5 degrees C, 2.7 mol/L NaCl, and 500 micromol/L H2O2 for 4 h, respectively. A green fluorescent protein gene- (gfp) labelled derivative of the Moraxella strain was used to examine the stress-resistant responses of the bacterium in natural river water microcosms. The carbon-starved gfp-labelled Moraxella strain also showed stress-resistant responses against heat, osmotic, and oxidative stresses in the river water samples. Despite the stress-tolerant capability of the carbon-starved gfp-labelled Moraxella cells, they did not exhibit any survival advantage over their non-starved counterparts when inoculated into river water microcosms and incubated at 10 and 22 degrees C for 14 d.

Transfer of the misnamed [Alysiella] sp. IAM 14971 (=ATCC 29468) to the genus Moraxella as Moraxella oblonga sp. nov.

Phylogenetic analysis based on 16S rRNA gene sequences revealed that [Alysiella] sp. IAM 14971 (=ATCC 29468) is closely related to the genus Moraxella of the gamma-Proteobacteria (96-97 % similarity). The newly obtained phenotypic data also indicate that [Alysiella] sp. IAM 14971 is distinct from the genus Alysiella and similar to the genus Moraxella. On the basis of these results, the strain should be classified in the genus Moraxella, as Moraxella oblonga sp. nov. The type strain is IAM 14971T (=ATCC 29468T).

Characterization of the first molluscicidal lipopolysaccharide from Moraxella osloensis.

Moraxella osloensis is a bacterium that is mutualistically associated with Phasmarhabditis hermaphrodita, a nematode that has potential for the biocontrol of mollusk pests, especially the slug Deroceras reticulatum. We discovered that purified M. osloensis lipopolysaccharide (LPS) possesses a lethal toxicity to D. reticulatum when administered by injection but no contact or oral toxicity to this slug. The toxicity of the LPS resides in the lipid A moiety. M. osloensis LPS was semiquantitated at 6 x 10(7) endotoxin units per mg. The LPS is a rough-type LPS with an estimated molecular weight of 5,300. Coinjection of galactosamine with the LPS increased the LPS's toxicity to the slug two- to four-fold. The galactosamine-induced sensitization of the slug to the LPS was reversed completely by uridine.

A high-molecular-weight outer membrane protein of Xanthomonas oryzae pv. oryzae exhibits similarity to non-fimbrial adhesins of animal pathogenic bacteria and is required for optimum virulence.

Transposon insertions in a novel 3.798 kb open reading frame (ORF) of the rice pathogen, Xanthomonas oryzae pv. oryzae (Xoo) cause virulence deficiency and altered colony/lawn morphology. This ORF encodes a protein, XadA, of 1,265 amino acids that exhibits significant similarity to non-fimbrial adhesins of animal pathogenic bacteria such as Yersinia YadA and Moraxella UspA1. An interesting feature is that the YadA similarity region is repeated six times within the XadA sequence and encompasses almost the entire length of the protein. Anti-XadA antibodies identified a 110 kDa outer membrane protein that was sensitive to protease treatment of whole cells. XadA expression is induced in minimal medium. Homology modelling suggests that XadA adopts a beta-helix conformation-like pertactin, a non-fimbrial adhesin of Bordetella pertussis. This work is the first characterization of a non-fimbrial adhesin-like molecule in a plant pathogenic bacterium. It extends our knowledge about the repertoire of homologous virulence factors that are deployed by animal and plant pathogenic bacteria to include functions potentially involved in adhesion.

Endotoxin activity of Moraxella osloensis against the grey garden slug, Deroceras reticulatum.

Moraxella osloensis is a gram-negative bacterium associated with Phasmarhabditis hermaphrodita, a slug-parasitic nematode that has prospects for biological control of mollusk pests, especially the grey garden slug, Deroceras reticulatum. This bacterium-feeding nematode acts as a vector that transports M. osloensis into the shell cavity of the slug, and the bacterium is the killing agent in the nematode-bacterium complex. We discovered that M. osloensis produces an endotoxin(s), which is tolerant to heat and protease treatments and kills the slug after injection into the shell cavity. Washed or broken cells treated with penicillin and streptomycin from 3-day M. osloensis cultures were more pathogenic than similar cells from 2-day M. osloensis cultures. However, heat and protease treatments and 2 days of storage at 22 degrees C increased the endotoxin activity of the young broken cells but not the endotoxin activity of the young washed cells treated with the antibiotics. This suggests that there may be a proteinaceous substance(s) that is structurally associated with the endotoxin(s) and masks its toxicity in the young bacterial cells. Moreover, 2 days of storage of the young washed bacterial cells at 22 degrees C enhanced their endotoxin activity if they were not treated with the antibiotics. Furthermore, purified lipopolysaccharide (LPS) from the 3-day M. osloensis cultures was toxic to slugs, with an estimated 50% lethal dose of 48 microg per slug, thus demonstrating that the LPS of M. osloensis is an endotoxin that is active against D. reticulatum. This appears to be the first report of a biological toxin that is active against mollusks.

The use of silica gel prepared by sol-gel method and polyurethane foam as microbial carriers in the continuous degradation of phenol.

A mixed microbial culture was immobilized by entrapment into silica gel (SG) and entrapment/ adsorption on polyurethane foam (PU) and ceramic foam. The phenol degradation performance of the SG biocatalyst was studied in a packed-bed reactor (PBR), packed-bed reactor with ceramic foam (PBRC) and fluidized-bed reactor (FBR). In continuous experiments the maximum degradation rate of phenol (q(s)max) decreased in the order: PBRC (598 mg l(-1) h(-1)) > PBR (PU, 471 mg l(-1)h(-1)) > PBR(SG, 394 mg l(-1) h(-1)) > FBR (PU, 161 mg l(-1) h(-1)) > FBR (SG, 91 mg l(-1) h(-1)). The long-term use of the SG biocatalyst in continuous phenol degradation resulted in the formation of a 100-200 microm thick layer with a high cell density on the surface of the gel particles. The abrasion of the surface layer in the FBR contributed to the poor degradation performance of this reactor configuration. Coating the ceramic foam with a layer of cells immobilized in colloidal SiO2 enhanced the phenol degradation efficiency during the first 3 days of the PBRC operation, in comparison with untreated ceramic packing.

Using a green fluorescent protein gene-labeled p-nitrophenol-degrading Moraxella strain to examine the protective effect of alginate encapsulation against protozoan grazing.

A gfp-labeled p-nitrophenol-degrading Moraxella strain G21 was used to study grazing of a Tetrahymena thermophila strain in liquid medium. This allowed visualization of the feeding process. Under an epifluorescent microscope, individual G21 fluorescent cells could be seen in vacuoles within the protozoans. Most of the G21 cells appeared to be lysed by T. thermophila and green fluorescent protein released from the bacteria yielded brightly fluorescent food vacuoles inside the protozoans, Examination of population dynamics of a mixed culture of T. thermophila and Moraxella sp. G21 showed that the protozoan reduced the bacterial density from 7.6 to 5.8 log CFU/ml in 2 days. Encapsulating the bacteria in alginate prevented grazing by the protozoans and the density of G21 cells in the beads increased steadily from about 8.3 to 8.9 log CFU/ml in 15 days regardless of the presence of the protozoans.

Formation of amino acid (L-leucine, L-phenylalanine) derived volatile flavour compounds by Moraxella phenylpyruvica and Staphylococcus xylosus in cured meat model systems.

A bacterial strain isolated from Danish immersion curing brine, Moraxella phenylpyruvica 0100, and a commercial meat starter culture, Staphylococcus xylosus DD34, were tested for their ability to form characteristic volatile compounds in minimal medium with the added amino acid L-leucine or L-phenylalanine under different environmental conditions (pH 5.5 and 6.0; 0 and 210 ppm nitrate; pre-incubation with and without agitation) and compared with respect to their ability to form volatile compounds in cured meat extracts and vacuum-packed cured meat cuts. The characteristic cured meat aroma precursors/compounds 3-methylbutanal and 3-methylbutanol were found to be formed in cured meat extracts and vacuum-packed cured meat cuts inoculated with M. phenylpyruvica. These volatiles are most probably formed by metabolic conversion of the amino acid L-leucine by M. phenylpyruvica, as they were also produced in minimal media with added L-leucine inoculated with this organism. The characteristic L-phenylalanine derived compound, benzaldehyde, formed by M. phenylpyruvica in minimal medium in the presence of nitrate (210 ppm), was not produced in any noticeable amount in cured meat extracts or vacuum-packed cured meat inoculated with M. phenylpyruvica. In contrast, benzacetaldehyde, which has been described as a possible metabolic product of the microbial conversion of L-phenylalanine, was found to be a characteristic volatile compound formed in cured meat extracts and vacuum-packed cured meat inoculated with M. phenylpyruvica, indicating an alternative metabolic pathway for L-phenylalanine by this organism in a cured meat environment. Even though S. xylosus was able to form volatile compounds characteristic of cured meats (3-methylbutanal, 3-methylbutanol) in minimal media with added L-leucine, this bacterial strain seemed not to be able to produce these characteristic volatiles in the studied cured meat systems. The present data imply that M. phenylpyruvica, in particular, is a potential meat starter for ensuring superior flavour development in cured meat.

Green fluorescent protein as a visual marker in a p-nitrophenol degrading Moraxella sp.

The green fluorescent protein gene (gfp) was introduced into a p-nitrophenol-metabolizing strain of Moraxella sp. by chromosomal integration. The gfp-marked transformants, designated Moraxella sp. strains G21 and G25, exhibited green fluorescence under UV light. Molecular characterization by PCR and Southern hybridization showed the presence of gfp in both transformants. Both transformants and the parent strain degraded 720 microM of p-nitrophenol with nitrite release within 4 h after inoculation in minimal medium supplemented with yeast extract. Transformants degraded up to 1440 microM p-nitrophenol and mineralized about 60% of 720 microM p-nitrophenol, both in broth and in soil, to the same extent as the parent strain. Insertion of gfp did not adversely affect the expression of p-nitrophenol-degrading genes in the transformants. Survival studies indicated that individual green fluorescent colonies of transformants can be detected up to 2 weeks after inoculation in soil. These marked strains could be of value in studies on microbial survival in the environment.