Two unusual chlorocatechol catabolic gene clusters in <Emphasis Type="Italic">Sphingomonas</Emphasis> sp. TFD44

The genes responsible for the degradation of 2,4-dichlorophenoxyacetate (2,4-D) by -Proteobacteria have previously been difficult to detect by using gene probes or polymerase chain reaction (PCR) primers. PCR products of the chlorocatechol 1,2-dioxygenase gene, tfdC, now allowed cloning of two chlorocatechol gene clusters from the Sphingomonas sp. strain TFD44. Sequence characterization showed that the first cluster, tfdD,RFCE, comprises all the genes necessary for the conversion of 3,5-dichlorocatechol to 3-oxoadipate, including a presumed regulatory gene, tfdR, of the LysR-type family. The second gene cluster, tfdC2E2F2, is incomplete and appears to lack a chloromuconate cycloisomerase gene and a regulatory gene. Purification and N-terminal sequencing of selected enzymes suggests that at least representatives of both gene clusters (TfdD of cluster 1 and TfdC2 of cluster 2) are induced during the growth of strain TFD44 with 2,4-D. A mutant constructed to contain an insertion in the chloromuconate cycloisomerase gene tfdD still was able to grow with 2,4-D, but more slowly and with a longer lag phase. This, and the detection of additional activity peaks during protein purification suggest that strain TFD44 harbors at least another chloromuconate cycloisomerase gene. The sequence of the tfdCE region was almost identical to that of a partially characterized chlorocatechol catabolic gene cluster of Sphingomonas herbicidovorans MH, whereas the sequence of the tfdC2E2F2 cluster was different. The similarity of the predicted proteins of the tfdD,RFCE and tfdC2E2F2 clusters to known sequences of other Proteobacteria in the database ranged from 42 to 61% identical positions for the first cluster and from 45.5 to 58% identical positions for the second cluster. Between both clusters, the similarities of their predicted proteins ranged from 44.5 to 64% identical positions. Thus, both clusters (together with those of S. herbicidovorans MH) represent deep-branching lines in the respective dendrograms, and the sequence information will help future primer design for the detection of corresponding genes in the environment.     

Detection of "<Emphasis Type="Italic">Rickettsia</Emphasis> sp. strain Uilenbergi" and "<Emphasis Type="Italic">Rickettsia</Emphasis> sp. strain Davousti" in <Emphasis Type="Italic">Amblyomma tholloni</Emphasis> ticks from elephants in Africa

Background  

To date, 6 tick-borne rickettsiae pathogenic for humans are known to occur in Africa and 4 of them were first identified in ticks before being recognized as human pathogens.     

Genetic and biochemical analyses of chromosome and plasmid gene homologues encoding ICL and ArCP domains in <Emphasis Type="Italic">Vibrio</Emphasis><Emphasis Type="Italic">anguillarum</Emphasis> strain 775

Anguibactin, the siderophore produced by Vibrio anguillarum 775 is synthesized from 2,3-dihydroxybenzoic acid (DHBA), cysteine and hydroxyhistamine via a nonribosomal peptide synthetase (NRPS) mechanism. Most of the genes encoding anguibactin biosynthetic proteins are harbored by the pJM1 plasmid. In this work we report the identification of a homologue of the plasmid-encoded angB on the chromosome of strain 775. The product of both genes harbor an isochorismate lyase (ICL) domain that converts isochorismic acid to 2,3-dihydro-2,3-dihydroxybenzoic acid, one of the steps of DHBA synthesis. We show in this work that both ICL domains are functional in the production of DHBA in V. anguillarum as well as in E. coli. Substitution by alanine of the aspartic acid residue in the active site of both ICL domains completely abolishes their isochorismate lyase activity in vivo. The two proteins also carry an aryl carrier protein (ArCP) domain. In contrast with the ICL domains only the plasmid encoded ArCP can participate in anguibactin production as determined by complementation analyses and site-directed mutagenesis in the active site of the plasmid encoded protein, S248A. The site-directed mutants, D37A in the ICL domain and S248A in the ArCP domain of the plasmid encoded AngB were also tested in vitro and clearly show the importance of each residue for the domain function and that each domain operates independently.     

Microbial degradation and metabolic pathway of pyridine by a <Emphasis Type="Italic">Paracoccus</Emphasis> sp. strain BW001

A bacterial strain using pyridine as sole carbon, nitrogen and energy source was isolated from the activated sludge of a coking wastewater treatment plant. By means of morphologic observation, physiological characteristics study and 16S rRNA gene sequence analysis, the strain was identified as the species of Paracoccus. The strain could degrade 2,614 mg l⁻¹ of pyridine completely within 49.5 h. Experiment designed to track the metabolic pathway showed that pyridine ring was cleaved between the C₂ and N, then the mineralization of the carbonous intermediate products may comply with the early proposed pathway and the transformation of the nitrogen may proceed on a new pathway of simultaneous heterotrophic nitrification and aerobic denitrification. During the degradation, NH₃-N occurred and increased along with the decrease of pyridine in the solution; but the total nitrogen decreased steadily and equaled to the quantity of NH₃-N when pyridine was degraded completely. Adding glucose into the medium as the extra carbon source would expedite the biodegradation of pyridine and the transformation of the nitrogen. The fragments of nirS gene and nosZ gene were amplified which implied that the BW001 had the potential abilities to reduce NO₂ ⁻ to NO and/or N₂O, and then to N₂.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Taxonomic characterization of <Emphasis Type="Italic">Haloferax</Emphasis> sp. ("<Emphasis Type="Italic">H. alicantei</Emphasis>") strain Aa 2.2: description of <Emphasis Type="Italic">Haloferax lucentensis</Emphasis> sp. nov.

An extremely halophilic archaeon, previously named as Haloferax sp. strain Aa 2.2 or "Haloferax alicantei" that has been extensively used for genetic studies with halobacteria, was taxonomically characterized by using phenotypic tests (including morphological, physiological, biochemical and nutritional features), DNA-DNA hybridization and 16S rRNA sequence phylogenetic analysis. This organism was isolated in 1986 by Torreblanca et al. from a pond of a Spanish saltern located in Alicante. The cells were pleomorphic, Gram negative and grew optimally at 25% NaCl. The polar lipid composition was similar to that of species of the genus Haloferax. The DNA G+C content of this strain was 64.5 mol%. Phylogenetic analysis based on 16S rRNA sequence comparison confirmed that this archaeon is a member of the genus Haloferax and was most closely related to Haloferax volcanii. DNA-DNA hybridization between strain Aa 2.2 and the type strain of all named species of the genus Haloferax revealed low levels of relatedness (25-2%), supporting the placement of this organism in a new species. On the basis of the phenotypic characteristics, molecular data and phylogenetic analysis we propose to name strain Aa 2.2 as a new species, Haloferax lucentensis sp. nov. The type strain is Aa 2.2 (=JCM 9276=NCIMB 13854=CIP 107410=DSM 14919=CECT 5871=CCM 7023).     

Phylogenetic analyses of <Emphasis Type="Italic">Uromyces viciae-fabae</Emphasis> and its varieties on <Emphasis Type="Italic">Vicia</Emphasis>, <Emphasis Type="Italic">Lathyrus</Emphasis>, and <Emphasis Type="Italic">Pisum</Emphasis> in Japan

A pea rust fungus, Uromyces viciae-fabae, has been classified into two varieties, var. viciae-fabae and var. orobi, based on differences in urediniospore wall thickness and putative host specificity in Japan. In principal component analyses, morphological features of urediniospores and teliospores of 94 rust specimens from Vicia, Lathyrus, and Pisum did not show definite host-specific morphological groups. In molecular analyses, 23 Uromyces specimens from Vicia, Lathyrus, and Pisum formed a single genetic clade based on D1/D2 and ITS regions. Four isolates of U. viciae-fabae from V. cracca and V. unijuga could infect and sporulate on P. sativum. These results suggest that U. viciae-fabae populations on different host plants are not biologically differentiated into groups that can be recognized as varieties.Contribution no. 184, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

Dehalorespiration with hexachlorobenzene and pentachlorobenzene by<Emphasis Type="Italic"> Dehalococcoides</Emphasis> sp. strain CBDB1

The chlororespiring anaerobe Dehalococcoides sp. strain CBDB1 used hexachlorobenzene and pentachlorobenzene as electron acceptors in an energy-conserving process with hydrogen as electron donor. Previous attempts to grow Dehalococcoides sp. strain CBDB1 with hexachlorobenzene or pentachlorobenzene as electron acceptors failed if these compounds were provided as solutions in hexadecane. However, Dehalococcoides sp. strain CBDB1 was able to grow with hexachlorobenzene or pentachlorobenzene when added in crystalline form directly to cultures. Growth of Dehalococcoides sp. strain CBDB1 by dehalorespiration resulted in a growth yield (Y) of 2.1±0.24 g protein/mol Cl^– released with hexachlorobenzene as electron acceptor; with pentachlorobenzene, the growth yield was 2.9±0.15 g/mol Cl^–. Hexachlorobenzene was reductively dechlorinated to pentachlorobenzene, which was converted to a mixture of 1,2,3,5- and 1,2,4,5-tetrachlorobenzene. Formation of 1,2,3,4-tetrachlorobenzene was not detected. The final end-products of hexachlorobenzene and pentachlorobenzene dechlorination were 1,3,5-trichlorobenzene, 1,3- and 1,4-dichlorobenzene, which were formed in a ratio of about 3:2:5. As reported previously, Dehalococcoides sp. strain CBDB1 converted 1,2,3,5-tetrachlorobenzene exclusively to 1,3,5-trichlorobenzene, and 1,2,4,5-tetrachlorobenzene exclusively to 1,2,4-trichlorobenzene. The organism therefore catalyzes two different pathways to dechlorinate highly chlorinated benzenes. In the route leading to 1,3,5-trichlorobenzene, only doubly flanked chlorine substituents were removed, while in the route leading to 1,3-and 1,4-dichlorobenzene via 1,2,4-trichlorobenzene singly flanked chlorine substituents were also removed. Reductive dehalogenase activity measurements using whole cells pregrown with different chlorobenzene congeners as electron acceptors indicated that different reductive dehalogenases might be induced by the different electron acceptors. To our knowledge, this is the first report describing reductive dechlorination of hexachlorobenzene and pentachlorobenzene via dehalorespiration by a pure bacterial culture.     

Mercury Tolerance of Thermophilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. and <Emphasis Type="Italic">Ureibacillus</Emphasis> sp

Although resistance of microorganisms to Hg(II) salts has been widely investigated and resistant strains have been reported from many eubacterial genera, there are few reports of mercuric ion resistance in extremophilic microorganisms. Moderately thermophilic mercury resistant bacteria were selected by growth at 62 °C on Luria agar containing HgCl₂. Sequence analysis of 16S rRNA genes of two isolates showed the closest matches to be with Bacillus pallidus and Ureibacillus thermosphaericus. Minimum inhibitory concentration (MIC) values for HgCl₂ were 80 μg/ml and 30 μg/ml for these isolates, respectively, compared to 10 μg/ml for B. pallidus H12 DSM3670, a mercury-sensitive control. The best-characterised mercury-resistant Bacillus strain, B. cereus RC607, had an MIC of 60 μg/ml. The new isolates had negligible mercuric reductase activity but removed Hg from the medium by the formation of a black precipitate, identified as HgS by X-ray powder diffraction analysis. No volatile H₂S was detected in the headspace of cultures in the absence or presence of Hg²⁺, and it is suggested that a new mechanism of Hg tolerance, based on the production of non-volatile thiol species, may have potential for decontamination of solutions containing Hg²⁺ without production of toxic volatile H₂S.     

<Emphasis Type="Italic">Kretzschmaria varians</Emphasis> sp. nov., <Emphasis Type="Italic">Xylaria coremiifera</Emphasis> sp. nov. and <Emphasis Type="Italic">Xylaria umbonata</Emphasis> sp. nov. from Costa Rica

Kretzschmaria varians, a species apparently related to K. micropus, is described as new. It is distinguished primarily by having asci with 2 to 8 ascospores with inconstant germination slit length and remains of synnemata on stromata and surrounding substrate. Xylaria coremiifera, described here as new, bears small fragile coremia on pulvinate stromata and the surrounding substrate. Asci often have fewer than 8 ascospores, most frequently 4. Xylaria umbonata, described here as new, produces perithecia around a central umbo that appears to be the remains of a synnema. Ascospores have long spiralling germination slits.     

Biodegradation of malathion by <Emphasis Type="Italic">Brevibacillus</Emphasis> sp. strain KB2 and <Emphasis Type="Italic">Bacillus cereus</Emphasis> strain PU

We report here the degradation of a pesticide, malathion, by Brevibacillus sp. strain KB2 and Bacillus cereus strain PU, isolated from soil samples collected from malathion contaminated field and an army firing range respectively. Both the strains were cultured in the presence of malathion under aerobic and energy-limiting conditions. Both strains grew well in the medium having malathion concentration up to 0.15%. Reverse phase HPLC–UV analysis indicated that Strain KB2 was able to degrade 72.20% of malaoxon (an analogue of malathion) and 36.22% of malathion, while strain PU degraded 87.40% of malaoxon and 49.31% of malathion, after 7 days of incubation. The metabolites mal-monocarboxylic acid and mal-dicarboxylic acid were identified by Gas chromatography/mass spectrometry. The factors affecting biodegradation efficiency were investigated and effect of malathion concentration on degradation rate was also determined. The strain was analyzed for carboxylesterase activity and maximum activity 210 ± 2.5 U ml⁻¹ and 270 U ± 2.7 ml⁻¹ was observed for strains KB2 and PU, respectively. Cloning and sequencing of putative malathion degrading carboxylesterase gene was done using primers based PCR approach.     

Studies on hydrogenase activity and chlorobenzene respiration in <Emphasis Type="Italic">Dehalococcoides</Emphasis> sp. strain CBDB1

Hydrogen oxidation and electron transport were studied in the chlorobenzene-utilizing anaerobe Dehalococcoides sp. strain CBDB1. While Cu²⁺ and Hg²⁺ ions irreversibly inhibited hydrogenase activity in intact cells, Ni²⁺ ions inhibited reversibly. About 80% of the initial hydrogenase activity was inactivated within 30 s when the cells were exposed to air. In contrast, hydrogenase was active at a redox potential of +10 mV when this redox potential was established anoxically with a redox indicator. Viologen dyes served both as electron acceptor for hydrogenase and electron donor for the dehalogenase. A menaquinone analogue, 2,3-dimethyl 1,4-naphthoquinone, served neither as electron acceptor for the hydrogenase nor as electron donor for the dehalogenase. In addition, the menaquinone antagonist 2-n-heptyl-4-hydroxyquinoline-N-oxide had no effect on dechlorination catalyzed by cell suspensions or isolated membranes with hydrogen as electron donor, lending further support to the notion that menaquinone is not involved in electron transport. The ionophores tetrachlorosalicylanilide and carbonylcyanide m-chlorophenylhydrazone did not inhibit dechlorination by cell suspensions, indicating that strain CBDB1 does not require reverse electron transport. The ATP-synthase inhibitor N,N-dicyclohexylcarbodiimide inhibited the dechlorination reaction with cell suspensions; however, the latter effect was partially relieved by the addition of tetrachlorosalicylanilide. 1,2,3,4-Tetrachlorobenzene strongly inhibited dechlorination of other chlorobenzenes by cell suspensions with hydrogen as electron donor, but it did not interfere with either hydrogenase or dehalogenase activity.     

Biosynthesis of silver nanoparticles by a new strain of <Emphasis Type="Italic">Streptomyces</Emphasis> sp. compared with <Emphasis Type="Italic">Aspergillus</Emphasis><Emphasis Type="Italic">fumigatus</Emphasis>

Locally isolated strains of a thermoalkalotolerant Streptomyces sp. and Aspergillus fumigatus were used for the in vitro biosynthesis of silver nanoparticles from AgNO₃ solutions. An autolysed cell-free culture filtrate from each strain was used, indicating that the formation mechanism depends on intra-cellular components for both organisms, since culture broths had no significant nanoparticle formation potential. Nanoparticle formation was indicated by a change of the solution from colourless or light brown to dark brown after 24 h or more, and UV–visible spectroscopy and x-ray diffraction analysis confirmed the formation by both organisms. The initial formation kinetics were faster with Aspergillus, but formation continued for a longer period with Streptomyces, resulting in higher concentrations after 48 h. Transmission electron microscope images revealed well dispersed nanoparticles with diameters ranging from 15 to 45 nm from A. fumigatus, while those from Streptomyces sp. had a narrower size distribution of 15–25 nm. The higher productivity and preferred narrower size distribution of Streptomyces, together with its well established industrial use, may make it the preferred choice for further optimization studies.     

Genetic relatedness of <Emphasis Type="Italic">Trichoderma</Emphasis> isolates antagonistic against <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f.sp. <Emphasis Type="Italic">dianthi</Emphasis> inflicting carnation wilt

Twenty-eight isolates of Trichoderma belonging to four different species were screened in vitro for their antagonistic ability against Fusarium oxysporum f.sp. dianthi causing carnation wilt. Three different levels of antagonism observed in dual plate assay were further confirmed by cell-free culture filtrate experiments. Isolates showing class I level of antagonism produced maximum lytic enzymes, chitinases and beta-1,3-glucanases. Genetic variability of 25 selected isolates was assessed by random amplified polymorphic DNA technique and the amplified products were correlated for their level of antagonism. Unweighed pair-group method with arithmetical averages cluster analysis revealed prominent inter-and intraspecific genetic variation among the isolates. Based on their genetic relationship, the isolates were mainly distributed into 3 major groups representing T. atroviride, T. pseudokoningii and T. harzianum, with 20-35% interspecific dissimilarity. However, the polymorphism shown by the isolates did not correlate to their level of antagonism.     

Polyhydroxybutyrate in <Emphasis Type="Italic">Rhizobium</Emphasis> and <Emphasis Type="Italic">Bradyrhizobium</Emphasis>: quantification and <Emphasis Type="Italic">phbC</Emphasis> gene expression

Polyhydroxyalkanoates (PHAs) are hydroxyalkanoate polymers that are produced and accumulate by many kinds of bacteria. These polymers act as an energy store for bacteria. Polyhydroxybutyrate (PHB) is the most studied polymer in the PHA family. These polymers have awakened interest in the environmental and industrial research areas because they are biodegradable and have thermoplastic qualities, like polypropylene. In this work, we analyzed the PHB production in Bradyrhizobium sp., Rhizobium leguminosarum bv. phaseoli, and Rhizobium huautlense cultured with two different carbon sources. We did biochemical quantification of PHB production during the three phases of growth. Moreover, these samples were used for RNA extraction and phbC gene expression analysis via real-time PCR. The bacteria showed different manner of growth, PHB accumulation and phbC gene expression when different quantity and quality of carbon sources were used. These results showed that under different growth media conditions, the growth and metabolism of different species of bacteria were influenced. These differences reflect the increase or decrease in PHB accumulation.