Development and relations of <Emphasis Type="Italic">Fusarium culmorum</Emphasis> and <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> in soil

The development of Fusarium culmorum and Pseudomonas fluorescens in soil, and the relations between them, were studied using membrane filters containing the fungus, the bacterium, or both microorganisms; the filters were incubated in soil. F. culmorum was identified by indirect immunofluorescence; the GUS-labeled strain was used to visualize P. fluorescens. It was found that F. culmorum introduced in soil can develop as a saprotroph, with the formation of mycelium, macroconidia, and a small amount of chlamydospores. Introduction of glucose and cellulose resulted in increased density of the F. culmorum mycelium and macroconidia. P. fluorescens suppressed the development of the F. culmorum mycelium in soil, but stimulated chlamydospore formation. Decreased mycelial density in the presence of P. fluorescens was more pronounced in soil without additions and less pronounced in the case of introduction of glucose or cellulose. F. culmorum had no effect on P. fluorescens growth in soil.     

Isolation of <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> species highly resistant to pentachlorobenzene

Polychlorinated aromatic compounds, including pentachlorobenzenes and hexachlorobenzenes, are recalcitrant industrial pollutants that cause adverse effects on living cells. In this paper, the isolation of Pseudomonas fluorescens species with high resistance to pentachlorobenzene (PeCB) is reported. It was found that, in contrast to its slightly negative effect on P. fluorescens growth, PeCB readily inhibited the cell growth of Serratia spp. and Escherichia coli strains, thus indicating that inhibition of bacterial growth by PeCB is species-dependent. Analysis of a P. fluorescens isolate revealed that the exposure to PeCB induced production of reactive oxygen species and led to an increase in the level of alkyl hydroperoxide reductase C (AhpC), an important enzyme enhancing the cell tolerance to organic hydroperoxides usually accumulated under oxidative stress. The putative mechanism conferring PeCB resistance to P. fluorescens and the potential use of P. fluorescens in bioremediation are discussed.     

Comparative genomic analysis of pyrene-degrading <Emphasis Type="Italic">Mycobacterium</Emphasis> species: Genomic islands and ring-hydroxylating dioxygenases involved in pyrene degradation

The genome sequences of two pyrene-degrading bacterial strains of Mycobacterium spp. PYR10 and PYR15, isolated from the estuarine wetland of the Han river, South Korea, were determined using the PacBio RS II sequencing platform. The complete genome of strain PYR15 was 6,037,017 bp in length with a GC content of 66.5%, and contained 5,933 protein-coding genes. The genome of strain PYR10 was 5,999,427 bp in length with a GC content of 67.7%, and contained 5,767 protein-coding genes. Based on the average nucleotide identity values, these strains were designated as M. gilvum PYR10 and M. pallens PYR15. A genomic comparison of these pyrene-degrading Mycobacterium strains with pyrene-non-degrading strains revealed that the genomes of pyrene-degrading strains possessed similar repertoires of ringhydroxylating dioxygenases (RHDs), including the pyrenehydroxylating dioxygenases encoded by nidA and nidA3, which could be readily distinguished from those of pyrenenon-degraders. Furthermore, genomic islands, containing catabolic gene clusters, were shared only among the pyrenedegrading Mycobacterium strains and these gene clusters contained RHD genes, including nidAB and nidA3B3. Our genome data should facilitate further studies on the evolution of the polycyclic aromatic hydrocarbon-degradation pathways in the genus Mycobacterium.     

<Emphasis Type="Italic">Lactobacillus fermentum</Emphasis> 90 TC-4 taxonomic status confirmation using whole genome sequencing and MALDI TOF mass spectrum

With the use of whole genome sequencing, the taxonomic status of Lactobacillus fermentum 90 TC-4 strain from Russian collections were studied. Complex analysis of phenotypical and genetic properties was conducted using phenotypic and molecular genetic methods. The main characteristics of the genome and biochemical activity profile of the strain were determined. A comparative analysis of the mass spectrum of ribosomal proteins of the strain, its biochemical properties, a fragment of 16S rRNA gene sequencing, and the entire genome revealed that the present strain belongs to the species L. fermentum, confirming its taxonomic status in accordance with modern taxonomy.     

Sulphur metabolism in Thiorhodaceae. III. Storage and turnover of thiosulphate sulphur inThiocapsa floridana andChromatium species

Thiocapsa floridana strain 1711 andChromatium strains 1211 and 1611 utilize sulphide, thiosulphate, and elementary sulphur as electron donors for growth; sulphite can be used only byChromatium strain 1611. In contrast to the other strains, thiosulphate utilization inChromatium strain 1211 is inducible and not constitutive: thiosulphate is consumed only after an induction period of about 20 hours. The turnover rate of different sulphur compounds is controlled by the CO₂ fixation rate. Using differently labeled³⁵S thiosulphates in short term experiments in a special stirred cuvette, it was shown that the maximum amount of stored intracellular sulphur depends on the strain as well as on the experimental conditions like pH and thiosulphate concentration. WhileChromatium strain 1211 showed a maximum storage of only 10% from sulphane-labeled thiosulphate at pH 6.7, and of 25.7% at pH 6.2,Thiocapsa floridana accumulated 75–90% of the radioactivity into the cells at pH 6.7. While in theChromatium strains the labeling of the cells remained at a constant level until all thiosulphate was consumed, inThiocapsa floridana a defined peak of radioactivity storage was obtained, followed by a steady but 3–4 times slower rate of excretion. With sulphonelabeled thiosulphate no significant accumulation of radioactivity occurred in the cells. During dark-incubation ofThiocapsa floridana (free of intracellular sulphur) in phosphate buffer, pH 6.5, with thiosulphate a production of sulphide could be measured while sulphite was not detected; no sulphide was produced by disrupted cells under the same conditions. The results obtained withThiocapsa floridana strongly support the concept of an initial cleavage of thiosulphate. The present observations do not allow a decision concerning the enzymatic mechanism of the cleavage itself.     

Resistance Mechanism in a Terbinafine-Resistant Strain of <Emphasis Type="Italic">Microsporum canis</Emphasis>

To clarify the terbinafine (TRF) resistance mechanism in a TRF-resistant strain of Microsporum canis, the expression of the pleiotropic drug resistance (PDR1), multidrug resistance (MDR1), MDR2 and MDR4 genes were investigated by real-time quantitative PCR (RT-qPCR) analysis, given the known interaction of the corresponding proteins with antifungals and with the efflux blocker FK506. The expression of the PDR1, MDR1, MDR2 and MDR4 genes was 2–4 times higher in the TRF-resistant strain grown in the presence of 0.14 µg/mL of TRF than in TRF-susceptible strains cultured in the absence of TRF. The TRF-resistant strain exhibited MICs of > 32 µg/mL for TRF alone; this resistance was attenuated to an MIC of 8 µg/mL in the presence of FK506, indicating that the TRF inhibitory concentration index value was < 0.75. The additive effect of the efflux blocker FK506 on TRF resistance was detected in the TRF-resistant strain. These results indicated that the TRF resistance in this strain reflects overexpression of genes encoding ABC transporter proteins.     

<Emphasis Type="Italic">Trueperella pyogenes</Emphasis> isolated from a brain abscess of an adult roebuck (<Emphasis Type="Italic">Capreolus capreolus</Emphasis>)

The present study was designed to characterize phenotypically and genotypically a Trueperella pyogenes strain isolated from a brain abscess of an adult roebuck (Capreolus capreolus). The species identity could be confirmed by phenotypical investigations, by MALDI-TOF MS analysis, and by sequencing the 16S ribosomal RNA (rRNA) gene, the 16S–23S rRNA intergenic spacer region (ISR); by sequencing the target genes rpoB, gap, and tuf; and by detection of T. pyogenes chaperonin-encoding gene cpn60 with a previously developed loop-mediated isothermal amplification (LAMP) assay. The T. pyogenes strain could additionally be characterized by PCR-mediated amplification of several known and putative virulence factor-encoding genes which revealed the presence of the genes plo encoding pyolysin and nanH and nanP encoding neuraminidases; the genes fimA, fimC, and fimE encoding the fimbrial subunits FimA, FimC, and FimE; and the gene cbpA encoding collagen-binding protein CbpA. The present data give a detailed characterization of a T. pyogenes strain isolated from a brain abscess of a roebuck. However, the route of infection of the roebuck remains unclear.     

Genome characteristics of the proteorhodopsin-containing marine flavobacterium <Emphasis Type="Italic">Polaribacter dokdonensis</Emphasis> DSW-5

Bacteria in the genus Polaribacter, belonging to the family Flavobacteriaceae, are typically isolated from marine environments. Polaribacter dokdonensis DSW-5, the type strain of the species, is a Gram-negative bacterium isolated from the East Sea of Korea. Whole genome shotgun sequencing was performed with the HiSeq 2000 platform and paired-end reads were generated at 188-fold coverage. The sequencing reads were assembled into two contigs with a total length of 3.08 Mb. The genome sequences of DSW-5 contain 2,776 proteincoding sequences and 41 RNA genes. Comparison of average nucleotide identities among six available Polaribacteria genomes including DSW-5 suggested that the DSW-5 genome is most similar to that of Polaribacter sp. MED152, which is a proteorhodopsin-containing marine bacterium. A phylogenomic analysis of the six Polaribacter strains and 245 Flavobacteriaceae bacteria confirmed a close relationship of the genus Polaribacter with Tenacibaculum and Kordia. DSW-5’s genome has a gene encoding proteorhodopsin and genes encoding 85 enzymes belonging to carbohydrate-active enzyme families and involved in polysaccharide degradation, which may play important roles in energy metabolism of the bacterium in the marine ecosystem. With genes for 238 CAZymes and 203 peptidases, DSW-5 has a relatively high number of degrading enzymes for its genome size suggesting its characteristics as a free-living marine heterotroph.     

In Vitro Selection and Identification of Potential Probiotics Isolated from the Gastrointestinal Tract of Nile Tilapia, <Emphasis Type="Italic">Oreochromis niloticus</Emphasis>

Fish gut bacteria can be used as probiotics for aquaculture. The aim of this study is to screen and identify beneficial probiotic bacteria from the gut of Nile tilapia, Oreochromis niloticus. Nine out of one hundred thirty-five isolates were non-pathogenic through intraperitoneal injection and had antibacterial activities with at least a strain from the five isolated fish pathogens, Aeromonas sobria, Aeromonas hydrophila, Pseudomonas aeruginosa, Pseudomonas putida, and Staphylococcus aureus. Further tests showed that such isolates can survive in the presence of high bile concentration (10%) and at different acidic pH values. A strains (14HT) was sensitive to all selected antibiotics, two strains were (9HT and 11HT) resistant to streptomycin and three strains (9HT, 11HT and 38HT) had resistance to two antibiotics. Four isolates (11HT, 33HT, 38HT and 41HT) had an amylase and a protease activities and one strain (47HT) showed only amylase activity. Based on 16S rRNA gene analysis, the isolated strains were identified as follows: Lactococcus lactis (8HT, 9HT, 11HT and 33HT); Enterococcus faecalis (14HT), Lysinibacillus sp. (38HT) and Citrobacter freundii (39HT, 41HT and 47HT).     

Genome sequencing of <Emphasis Type="Italic">Pediococcus acidilactici</Emphasis> (NRCC1), a novel isolate from dromedary camel (<Emphasis Type="Italic">Camelus dromedarius</Emphasis>) rumen fluid

The lactic acid bacterium Pediococcus acidilactici has recently been reported to help in treating constipation, diarrhea, relieving stress, and enhancing growth rate and immune response in humans, birds, fishes, and small animals. In the present study, we sequenced and analyzed the whole genome of P. acidilactici NRCC1, a novel isolate from rumen fluid of dromedary camel (Camelus dromedarius). The genome of P. acidilactici NRCC1 was assembled into 60 contigs, comprising 1,785,679 bp and 42.5% GC content. The 1705 CDS were predicted and annotated using the RAST server. The genome encodes numerous enzymes for utilization of different carbohydrates. It also harbors genes for antibiotic biosynthesis and many others which might confer probiotic properties. The comparative genome analysis with P. acidilactici DSM 20284 revealed some unique features in P. acidilactici NRCC1. Thus, the genome sequencing of P. acidilactici NRCC1 has opened up new horizons for further research in animal probiotics and feed supplements.     

Identification of the di-<Emphasis Type="Italic">n</Emphasis>-butyl phthalate-biodegrading strains and the biodegradation pathway in strain LMB-1

DNA isolated from a greenhouse soil (Nanjing, Jiangsu Province, China) was suitable for PCR amplification of gene segment coding for the 16S rRNA. Diverse PCR products were characterized by cloning and sequencing, and analysis of bacterial colonies showed the presence over 26 phyla. The most bacteria belonged to Proteobacteria, Actinobacteria, Gemmatimonadetes, Acidobacteria and Planctomycetes. Furthermore, after the enrichment procedure of DBP-degrading microorganisms, 4 strains were isolated from the soil sample with di-n-butyl phthalate (DBP) biodegradability, and they were identified to be Rhizobium sp., Streptomyces sp., Pseudomonas sp. and Acinetobacter sp. Analysis of the degradation products by LC-MS led to identification of metabolites of DBP in strain LMB-1 (identified as Rhizobium sp.) which suggests that DBP was degraded through β-oxidation, demethylation, de-esterification and cleavage of aromatic ring.     

Adaptive mutation related to cellulose producibility in <Emphasis Type="Italic">Komagataeibacter medellinensis</Emphasis> (<Emphasis Type="Italic">Gluconacetobacter xylinus</Emphasis>) NBRC 3288

Gluconacetobacter xylinus (formerly Acetobacter xylinum and presently Komagataeibacter medellinensis) is known to produce cellulose as a stable pellicle. However, it is also well known to lose this ability very easily. We investigated the on and off mechanisms of cellulose producibility in two independent cellulose-producing strains, R1 and R2. Both these strains were isolated through a repetitive static culture of a non-cellulose-producing K. medellinensis NBRC 3288 parental strain. Two cellulose synthase operons, types I and II, of this strain are truncated by the frameshift mutation in the bcsBI gene and transposon insertion in the bcsCII gene, respectively. The draft genome sequencing of R1 and R2 strains revealed that in both strains the bcsBI gene was restored by deletion of a nucleotide in its C-rich region. This result suggests that the mutations in the bcsBI gene are responsible for the on and off mechanism of cellulose producibility. When we looked at the genomic DNA sequences of other Komagataeibacter species, several non-cellulose-producing strains were found to contain similar defects in the type I and/or type II cellulose synthase operons. Furthermore, the phylogenetic relationship among cellulose synthase genes conserved in other bacterial species was analyzed. We observed that the cellulose genes in the Komagataeibacter shared sequence similarities with the γ-proteobacterial species but not with the α-proteobacteria and that the type I and type II operons could be diverged from a same ancestor in Komagataeibacter.     

Effect of acidic conditions on fatty acid composition and membrane fluidity of<Emphasis Type="Italic">Escherichia coli</Emphasis> strains isolated from Crescenza cheese

The effect of low pH on minimum pH, growth kinetics and adaptive response to acidity conditions were studied on nineEscherichia coli strains having food origin and two collection strains. Moreover, the effects of low pH on the modification of fatty acid (FA) composition and membrane fluidity of the strains were studied. Also the effects of a pre-adaptation to pH 5 on acidic resistance and FA composition were considered. The results showed the crucial role of unsaturated FA in acid adaptation ofE. coli. Cyclization and isomerization of membrane FA represented the most interesting response to acidic growth conditions in theE. coli strains considered. In particularcis/trans isomerization was shown to play an important role in modulating membrane composition and fluidity under acidic conditions. Only one strain was able to adapt to acidic stress increasing its ability to grow at low pH. It responded to pH decrease, conversely to each other strain, by increasing its membrane fluidity at the lowest pH values considered. The results obtained showed that the determination of membrane fluidity in intact whole cells by fluorescence polarisation can represent a useful tool to study instantaneous stress-induced membrane fluidity modifications.     

<Emphasis Type="Italic">Halomonas chromatireducens</Emphasis> sp. nov., a new denitrifying facultatively haloalkaliphilic bacterium from solonchak soil capable of aerobic chromate reduction

A heterotrophic bacterial strain AGD 8-3 capable of denitrification under extreme haloalkaline conditions was isolated from soda solonchak soils of the Kulunda steppe (Russia). The strain was classified within the genus Halomonas. According to the results of 16S rRNA gene sequencing, Halomonas axialensis, H. meridiana, and H. aquamarina are most closely related to strain AGD 8-3 (96.6% similarity). Similar to other members of the genus, the strain can grow within a wide range of salinity and pH. The strain was found to be capable of aerobic reduction of chromate and selenite on mineral media at 160 g/l salinity and pH 9.5–10. The relatively low level of phylogenetic similarity and the phenotypic characteristics supported classification of strain AGD 8-3 as a new species Halomonas chromatireducens.     

Genome sequencing and analysis of <Emphasis Type="Italic">Kloeckera apiculata</Emphasis> strain 34-9, a biocontrol agent against postharvest pathogens in citrus

Kloeckera apiculata, as the anamorphic state of Hanseniaspora uvarum from the Ascomycota phylum, plays an important role in the inhibition of fungal diseases in plants and spontaneous wine fermentation. This study was performed to sequence and analyze the whole genome of K. apiculata strain 34-9; This analysis provides further genomic features and assists functional research. The complete genome was determined using an Illumina HiSeq 2000 system applying paired-end and mate-pair methods to construct four reads libraries. The data assembly of all the reads resulted in a total genome size of 8.1 Mb, including 106 contigs, which were assembled into 41 scaffolds with a 31.95 % G+C content and a 234X sequence coverage. The performance of the gene prediction and functional annotation revealed that 2724 of 3786 protein-coding genes matched the KOG database, and 1127 genes were classified into GO categories. Further genome features analyses found 1066 microsatellite sites, 71 tRNAs, 3 rRNAs and 3 microRNAs in the genomic DNA. A prediction of the metabolic pathways identified potentially crucial genes for explaining the phenylalanine pathway involved in biocontrol. Comparisons with the typical yeasts Lachancea thermotolerans, Kluyveromyces lactis and Saccharomyces cerevisiae revealed the particularity and difference of K. apiculata strain 34-9. The genome alignments among Hanseniaspora vineae T02/19AF, K. apiculata DSM 2768 and 34-9 indicated numerous homologous regions distributed over the genomes between strain DSM2768 and 34-9. A SSR analysis identified that mono- and tri- nucleotide repeat types were more abundant in all six types, likely affecting the evolution of K. apiculata.     

Chromosomal Sil system contributes to silver resistance in <Emphasis Type="Italic">E. coli</Emphasis> ATCC 8739

The rise of antibiotic resistance in pathogenic bacteria is endangering the efficacy of antibiotics, which consequently results in greater use of silver as a biocide. Chromosomal mapping of the Cus system or plasmid encoded Sil system and their relationship with silver resistance was studied for several gram-negative bacteria. However, only few reports investigated silver detoxification mediated by the Sil system integrated in Escherichia coli chromosome. Accordingly, this work aimed to study the Sil system in E. coli ATCC 8739 and to produce evidence for its role in silver resistance development. Silver resistance was induced in E. coli ATCC 8739 by stepwise passage in culture media containing increasing concentrations of AgNO₃. The published genome of E. coli ATCC 8739 contains a region showing strong homology to the Sil system genes. The role of this region in E. coli ATCC 8739 was assessed by monitoring the expression of silC upon silver stress, which resulted in a 350-fold increased expression. De novo sequencing of the whole genome of a silver resistant strain derived from E. coli ATCC 8739 revealed mutations in ORFs putative for SilR and CusR. The silver resistant strain (E. coli AgNO₃R) showed constitutive expression of silC which posed a cost of fitness resulting in retarded growth. Furthermore, E. coli AgNO₃R exhibited cross-resistance to ciprofloxacin and a slightly increased tolerance to ampicillin. This study demonstrates that E. coli is able to develop resistance to silver, which may pose a threat towards an effective use of silver compounds as antiseptics.