Genetic and phenotypic microdiversity of Ochrobactrum spp

The diversity of Ochrobactrum anthropi, Ochrobactrum intermedium, Ochrobactrum tritici and Ochrobactrum grignonense in agricultural soil and on the wheat rhizoplane was investigated. O. anthropi was isolated both from soil and from the rhizoplane, O. intermedium and grignonense only from bulk soil, and O. tritici only from the wheat rhizoplane. On the genetic level, the immunotrapped isolates and a number of strains from culture collection mainly of clinical origin were compared with rep-PCR profiling using BOX primers, and a subset of these isolates and strains using REP primers. The isolates clustered according to their species affiliation. There was no correlation between rep clusters of O. anthropi isolates and habitat (place of isolation). The genetic diversity of Ochrobactrum at the species level as well as microdiversity of O. anthropi (number of BOX groups) was higher in soil than on the rhizoplane. Similarity values from genetic rep-PCR profiles correlated positively with DNA-DNA reassociation percentages. Isolates with >80.7% similarity in BOX profile and >86.4% in rep profile clustered within the same species. Similarity analysis of rep-PCR profiles is hence an alternative to DNA-DNA hybridization as a genomic criterion for species delineation within the genus Ochrobactrum. We used the substrate utilization system BIOLOG-GN to compare the immunotrapped isolates on the phenetic level. For the isolates from bulk soil, substrate utilization versatility (number of utilized substrates) and substrate utilization capacity (mean conversion rate of substrates) were slightly but significantly higher than for the isolates from the rhizoplane. This trend was also seen using API 20E and 20NE systems. Plate counts of total bacteria and the number of immunotrapped Ochrobactrum isolates per gram dry weight were higher for the rhizoplane than for the soil samples. The results of genetic and phenotypic analyses indicated a 'rhizosphere effect'; the diversity and metabolic capacity of Ochrobactrum isolates were higher in bulk soil, and the population density was higher on the wheat rhizoplane.     

Endophytic colonization ability of two deep-water rice endophytes, Pantoea sp. and Ochrobactrum sp. using green fluorescent protein reporter

Colonization ability of the two endophytic bacteria, isolated from surface sterilized seeds of Jaisurya variety of deep-water rice viz., Pantoea sp. and Ochrobactrum sp., was compared after genetically tagging them with a constitutively expressing green fluorescent protein gene (gfp). Confocal laser scanning microscopy (CLSM) of hydroponically grown seedlings of Jaisurya rice, inoculated with gfp-tagged endophytes, revealed that both Pantoea sp. and Ochrobactrum sp. colonized the intercellular spaces in the root cortex when inoculated separately. Colonization by gfp-tagged Ochrobactrum sp. was severely inhibited when co-inoculated with an equal number (10(5) c.f.u. ml(-1)) of wild type Pantoea sp., but the converse was not true. Pantoea sp. was a more aggressive endophytic colonizer of its host than Ochrobactrum sp. The potential of using GFP reporter and CLSM as tools in evaluating competitive ability of colonization among endophytes is herewith demonstrated.     

Detection and characterization of bacteria from the potato rhizosphere degrading N-acyl-homoserine lactone

Quorum sensing plays a role in the regulation of soft rot diseases caused by the plant pathogenic bacterium Pectobacterium carotovorum subsp. carotovorum. The signal molecules involved in quorum sensing in P. carotovorum subsp. carotovorum belong to the group of N-acyl homoserine lactones (AHLs). In our study, we screened bacteria isolated from the potato rhizosphere for the ability to degrade AHLs produced by P. carotovorum subsp. carotovorum. Six isolates able to degrade AHLs were selected for further studies. According to 16S rDNA sequence analysis and fatty acid methyl ester profiling, the isolates belonged to the genera Ochrobactrum, Rhodococcus, Pseudomonas, Bacillus, and Delftia. For the genera Ochrobactrum and Delftia, for the first time AHL-degrading isolates were found. Data presented in this study revealed for the first time that Ochrobactrum sp. strain A44 showed the capacity to inactivate various synthetic AHL molecules; the substituted AHLs were inactivated with a lower efficiency than the unsubstituted AHLs. Compared with the other isolates, A44 was very effective in the degradation of AHLs produced by P. carotovorum subsp. carotovorum. It was verified by polymerase chain reaction, DNA-DNA hybridization, and a lactone ring reconstruction assay that Ochrobactrum sp. strain A44 did not possess AHL lactonase activity. AHL degradation in Ochrobactrum sp. strain A44 occurred intracellularly; it was not found in the culture supernatant. AHL-degrading activity of A44 was thermo sensitive. Experiments in planta revealed that Ochrobactrum sp. strain A44 significantly inhibited the maceration of potato tuber tissue. Since A44 did not produce antibiotics, the attenuation of the decay might be due to the quenching of quorum- sensing-regulated production of pectinolytic enzymes. The strain can potentially serve to control P. carotovorum subsp. carotovorum in potato.     

Degradation of phenanthrene via meta-cleavage of 2-hydroxy-1-naphthoic acid by Ochrobactrum sp. strain PWTJD

The present study describes the assimilation of phenanthrene by an aerobic bacterium, Ochrobactrum sp. strain PWTJD, isolated from municipal waste-contaminated soil sample utilizing phenanthrene as a sole source of carbon and energy. The isolate was identified as Ochrobactrum sp. based on the morphological, nutritional and biochemical characteristics as well as 16S rRNA gene sequence analysis. A combination of chromatographic analyses, oxygen uptake assay and enzymatic studies confirmed the degradation of phenanthrene by the strain PWTJD via 2-hydroxy-1-naphthoic acid, salicylic acid and catechol. The strain PWTJD could also utilize 2-hydroxy-1-naphthoic acid and salicylic acid, while the former was metabolized by a ferric-dependent meta-cleavage dioxygenase. In the lower pathway, salicylic acid was metabolized to catechol and was further degraded by catechol 2,3-dioxygenase to 2-hydroxymuconoaldehyde acid, ultimately leading to tricarboxylic acid cycle intermediates. This is the first report of the complete degradation of a polycyclic aromatic hydrocarbon molecule by Gram-negative Ochrobactrum sp. describing the involvement of the meta-cleavage pathway of 2-hydroxy-1-naphthoic acid in phenanthrene assimilation.     

A novel symbiotic nitrogen-fixing member of the Ochrobactrum clade isolated from root nodules of Acacia mangium

Ten strains of root nodule bacteria were isolated from the nodules of Acacia mangium grown in the Philippines and Thailand. Partial sequences (approx. 300 bp) of the 16S rRNA gene of each isolate were analyzed. The nucleotide sequences of strain DASA 35030 indicated high homology (>99%) with members of the genus Ochrobactrum in Brucellaceae, although the sequences of other isolates were homologous to those of two distinct genera Bradyrhizobium and Rhizobium. The strain DASA 35030 was strongly suggested to be a strain of Ochrobactrum by full length sequences of the 16S rRNA gene, fatty acids composition, G+C contents of the DNA, and other physiological characteristics. Strain DASA 35030 induced root nodules on A. mangium, A. albida and Paraserianthes falcataria. The nodules formed by strain DASA 35030 fixed nitrogen and the morphology of the nodules is the same as those of nodules formed by the other isolates. This is the first report that the strain of Ochrobactrum possesses complete symbiotic ability with Acacia.     

Specificity inversion of Ochrobactrum anthropi D-aminopeptidase to a D,D-carboxypeptidase with new penicillin binding activity by directed mutagenesis

The serine penicillin-recognizing proteins have been extensively studied. They show a wide range of substrate specificities accompanied by multidomain features. Their adaptation capacity has resulted in the emergence of pathogenic bacteria resistant to beta-lactam antibiotics. The most divergent enzymatic activities in this protein family are those of the Ochrobactrum anthropi D-aminopeptidase and of the Streptomyces R61 D,D-carboxypeptidase/transpeptidase. With the help of structural data, we have attempted to identify the factors responsible for this opposite specificity. A loop deletion mutant of the Ochrobactrum anthropi D-aminopeptidase lost its original activity in favor of a new penicillin-binding activity. D-aminopeptidase activity of the deletion mutant can be restored by complementation with another deletion mutant corresponding to the noncatalytic domain of the wild-type enzyme. By a second step site-directed mutagenesis, the specificity of the Ochrobactrum anthropi D-aminopeptidase was inverted to a D,D-carboxypeptidase specificity. These results imply a core enzyme with high diversity potential surrounded by specificity modulators. It is the first example of drastic specificity change in the serine penicillin-recognizing proteins. These results open new perspectives in the conception of new enzymes with nonnatural specificities. The structure/specificity relationship in the serine penicillin-recognizing proteins are discussed.     

Comparative sequence analysis of the internal transcribed spacer 1 of Ochrobactrum species

The internal 16S/23S rDNA (rrs/rrl) internal spacer region 1 (ITS1) of 54 Ochrobactrum strains and close relatives was analysed. Separation of ITS1 containing PCR products by gel-electrophoresis, DGGE, cloning and sequencing revealed ITS1 length and sequence heterogeneity. We found up to 5 different allelic ITS1 stretches within a single strain (Ochrobactrum intermedium LMG 3301T), and 2-3 different ITS1 alleles in O. tritici. Within ITS1, ITS1c, being part of the conserved double-stranded rrn processing stem dsPS1, produced the most reliable segment tree. The overall ITS1, ITS1c and rrs phylogenetic tree topologies were generally consistent, but there was evidence for horizontal rrn (segment) transfer in O. tritici LMG 2134 (formerly O. anthropi). Good correlations were found between ITS1, ITS1c and rrs sequence similarity and DNA-DNA hybridization values indicating that phylogenetic analysis of ITS1 and ITS1c both can be used to preliminarily deduce the phylogenetic affiliation if HGT was excluded. Strains sharing > 96.19% ITS1c (> 95.11% ITS1) similarity fell within a species, and < or = 68.42% ITS1c (< or = 70.33% ITS1) similarity outside a genus. Both ITS1 and ITS1c analysis resolved microdiversity more profoundly than rrs analysis and revealed clades (genomovars) within O. anthropi that were also produced in rep cluster analysis. There was no evidence for habitat-specific ITS1 genomovars within Ochrobactrum species. Diversity of Ochrobactrum was higher in soil than at the rhizoplane below and at the species level. Isolates from soil contained only 1 rrn type whereas isolates from human clinical, animal and rhizoplane specimens could contain more.     

Role of a serine-type d-alanyl-d-alanine carboxypeptidase on the survival of Ochrobactrum sp. 11a under ionic and hyperosmotic stress

The plant growth-promoting rhizobacterium, Ochrobactrum sp. 11a displays a high intrinsic salinity tolerance and has been used in this work to study the molecular basis of bacterial responses to high concentrations of NaCl. A collection of Ochrobactrum sp. 11a mutants was generated by Tn 5 -B21 mutagenesis and screened for sensitivity to salinity. One clone, designated PBP and unable to grow on glutamate mannitol salt agar medium supplemented with 300 mM NaCl was selected and further characterized. The PBP mutant carries a single transposon insertion in a gene showing a high degree of identity to the serine-type d -alanyl- d -alanine carboxypeptidase gene of Ochrobactrum anthropi . Interestingly, the expression of this gene was shown to be upregulated by salt in the PBP mutant. Moreover, evidence is presented for the requirement of the gene product for adaptation to high-salt conditions as well as to overcome the toxicity of LiCl, KCl, sucrose, polyethylene glycol (PEG), AlCl₃, CuSO₄, and ZnSO₄. In addition to the altered tolerance to both ionic and osmotic stresses, the PBP mutant exhibited changes in colony and cell morphology, exopolysaccharide production, and an increased sensitivity to detergents.     

Alkane utilization by Rhodococcus strain NTU-1 alone and in its natural association with Bacillus fusiformis L-1 and Ochrobactrum sp

Linear (n-hexadecane) and branched (pristane) alkanes were degraded by a mixed culture isolated from an oil-contaminated field. The degradation was accompanied by formation of biofloccules. The culture was composed of Rhodococcus strain NTU-1, Bacillus fusiformis L-1, and Ochrobactrum sp. Rhodococcus strain NTU-1 carried out the degradation of the alkane via a hydroxylase. Bacillus fusiformis L-1 and Ochrobactrum sp. did not degrade the alkanes but aided the flocculation by forming more rigid bacterial aggregates that enhanced the trapping of alkanes. In batch cultures, transformation and removal of the linear and branched alkanes was achieved within 66 h with more than 95% efficiency.     

利用细菌还原有毒Cr(Ⅵ)为Cr(Ⅲ)

Overthelastfewdecadesenvironmentalcontaminationwithheavymetalshasincreaseddrastically .Heavymetalsfoundinwastewatersareharmfultotheenvironmentandtheireffectsonbiolo gicalsystemareverysevere.Anefficientandcheaptreatmentfortheirremovalandreuseofspentmetalsfromwastewaterneedstobedeve loped .Theremovaloftoxicmetalsfromtheenvironmentbymi croorganismshaspotentialasaneffectivemeansofremediatingheavymetalswastes.Microbe basedtechnologiescanprovideanalternativetoconventionalmethodsformetalremoval[1 ] .…     

Genotypic and phenotypic similarity between bacteria of Brucella genus and Rhizobiaceae family as a basis for reconsideration of Brucella taxonomy

Accumulated data on assessment of genome of bacteria from Brucella genus and Rhizobiaceae family (results of sequencing, DNA-rRNA hybridization, 16S rRNA gene sequencing etc.) as well as their phenotypic characteristics (first of all, composition of cell fatty acids) were summarized. Data point to phylogenetic proximity of these bacteria and possibility to unite them in one Rhizobiaceae family together with the closest relatives of Brucella--first of all, with bacteria from Ochrobactrum genus). This seems to be more objective than recreation of Brucellaceae family (Rhizobiales order) with genera Brucella, Ochrobactrum and, possibly, others.     

Ochrobactrum tritici strain 5bvl1 - characterization of a Cr(VI)-resistant and Cr(VI)-reducing strain

Bacterial strain 5bvl1, isolated from a chromium-contaminated wastewater treatment plant and identified as Ochrobactrum tritici, was resistant to a broad range of antibiotics, to Cr(VI), Ni(II), Co(II), Cd(II), and Zn(II), and was able to grow in the presence of 5% NaCl and within the pH range 4-10. Characterization showed that strain 5bvl1 could be considered a halotolerant and alkalitolerant microorganism resistant to high concentrations of Cr(VI). This strain was able to grow aerobically in up to 10 mmolxL(-1) Cr(VI). Cr(VI) resistance was independent of sulphate concentration. Under aerobic conditions strain 5bvl1 was also able to reduce high Cr(VI) concentrations (up to 1.7 mmolxL(-1)). Increasing concentrations of Cr(VI) in the medium lowered the growth rate of strain 5bv11 but the reduction in growth rate could not be directly correlated with the amount of Cr(VI) reduced. Unlike the type strain, which was only able to reduce Cr(VI), strain 5bvl1 was resistant to Cr(VI) and able to reduce it. Moreover, in strain 5bvl1, the rate and extent of Cr(VI)-reduction were higher than in the other strains of the genus Ochrobactrum. Ochrobactrum strain 5bvl1 resists high Cr(VI) concentrations and has a high Cr(VI)-reducing ability, making it a valuable tool in bioremediation.     

DNA cloning and sequence analysis of chicken AFLP

Amplified fragment length polymorphisms (AFLP) have been shown to be useful for linkage mapping in chickens and other domestic animals. It is often desirable to convert AFLP bands to sequence-tagged site (STS) markers, in particular, so that AFLP-based linkage information can be integrated with recombinant DNA clone-based maps. Sixteen chicken AFLP bands were excised from gels, re-amplified, cloned and analysed. All inserts proved to be EcoRI-TaqI fragments, which suggests that unlabelled TaqI-TaqI AFLP fragments do not amplify well, and therefore do not significantly contaminate AFLP bands. For eight of the AFLP, the cloned fragment was used to probe blots of AFLP reaction fingerprints, confirming that the predominant DNA clone indeed contained the polymorphic fragment. Flanking regions of selected AFLP fragments were isolated using Vectorette cloning. The results obtained suggest that the these chicken AFLP most commonly arise from sequence polymorphism at or near the TaqI site.     

Evaluation of Ochrobactrum anthropi TRS-2 and its talc based formulation for enhancement of growth of tea plants and management of brown root rot disease

Aim: To evaluate Ochrobactrum anthropi TRS‐2 isolated from tea rhizosphere and its talc based formulation for growth promotion and management of brown root rot disease of tea. Methods and Results: Ochrobactrum anthropi TRS‐2, isolated from tea rhizosphere could solubilize phosphate, produce siderophore and IAA in vitro and also exhibited antifungal activity against six test pathogens. Application of an aqueous suspension of O. anthropi to the rhizosphere of nursery grown tea seedlings of five varieties of tea (TV‐18, T‐17, HV‐39, S‐449, UP‐3 and) led to enhanced growth of the treated plants, as evidenced by increase in height, in the number of shoots and number of leaves per shoot. Treatment with O. anthropi also decreased brown root rot of tea, caused by Phellinus noxius. Multifold increase in activities of chitinase, β‐1,3‐glucanase, peroxidase and phenylalanine ammonia lyase in tea plants was observed on application of O. anthropi to soil followed by inoculation with P. noxius. A concomitant increase in accumulation of phenolics was also obtained. Further, talc based formulation of O. anthropi was prepared and its survival determined every month up to a period of 12 months. Ochrobactrum anthropi could survive in the formulation up to a period of 9 months with a concentration of 7·0 log₁₀ CFU g^?1, after which there was a decline. Talc formulation was as effective as aqueous suspensions in both plant growth promotion and disease suppression. Conclusion: Ochrobactrum anthropi, either in aqueous suspension or as talc formulation induced growth of tea plants and suppressed brown root rot disease. It induced defense responses in tea plants. Significance and Impact of the Study: Ochrobactrum anthropi and its talc based formulation can be considered as an addition to available plant growth promoting rhizobacteria (PGPR) currently being used for field application. The present study offers a scope of utilizing this bacterium for growth promotion and disease management which would help in reduction of the use of chemicals in tea plantations.     

Effect of aeration on denitrification byOchrobactrum anthropi SY509

Aeration was found to affect the biological denitrification byOchrobactrum anthropi SY509. Although cell growth was vigorous under 1 vvm of aeration and an agitation speed of 400 rpm in a 3-L jar fermentor, almost no nitrate was removed. Yet under low agitation speeds (100, 200, and 300 rpm), denitrification occurred when the dissolved oxygen was exhausted shortly after the inoculation of the microorganism.Ochrobactrum anthropi SY509 was found to express highly active denitrifying enzymes under anaerobic conditions. The microorganism also synthesized denitrifying enzymes under aerobic conditions (1 vvm and 400 rpm), yet their activity was only 60% of the maximum level under anaerobic conditions and the nitrate removal efficiency was merely 15%. However, although the activities of the denitrifying enzymes were inhibited in the presence of oxygen, they were fully recovered when the conditions were switched to anaerobic conditions.     

AFLP analysis for examining genetic differences in cultivated strains and their single-spore isolates and for confirming successful crosses in <Emphasis Type="Italic">Agaricus blazei</Emphasis>

Amplified fragment length polymorphism (AFLP) analysis was used to examine genetic differences in Agaricus blazei cultivated strains and their single-spore isolates (SSIs). AFLP analysis with five primer combinations identified a total of 267 AFLP bands from nine cultivated strains (one from Brazil and eight from Japan), of which 165 were polymorphic between the nine strains. An AFLP data dendrogram grouped the eight Japanese strains, with the Brazilian strain acting as an outlier, suggesting that the Brazilian and Japanese strains are genetically quite different. Twelve SSIs derived from each of four cultivated strains were subjected to AFLP analysis. All the AFLP bands detected in the cultivated strains were also found in at least one SSI, but some unique bands were detected in SSIs. The total number of AFLP bands from individual SSIs was clearly less than those from their parental strains, and many of polymorphic AFLP bands from the parental strains segregated in SSIs at a ratio of 1 : 1, suggesting that the SSIs are homokaryotic. Distance values based on presence or absence of individual AFLP bands among SSIs from different strains were clearly higher than those among SSIs from a single strain. In addition, AFLP analysis was shown to be useful in confirming hybrid formation in crosses between SSIs.     

降解畜禽养殖废水污泥的优势菌株筛选及其性能研究

为了实现畜禽养殖废水处理过程中污泥的减量化,从不同样品中筛选得到8株污泥降解菌。以污泥作为唯一的碳源和能源来验证8株菌的污泥降解效果,最终得到3株污泥降解优势菌株,分别为嗜线虫沙雷氏菌Serratia nematodiphila H1、产吲哚金黄杆菌Chryseobacterium indologenes B4和苍白杆菌Ochrobactrum thiophenivorans D7。其中嗜线虫沙雷氏菌H1对污泥的可挥发性悬浮固体(volatile suspended solids,VSS)去除率最高,达52. 9%,比对照组的41. 6%提高27. 2%;产吲哚金黄杆菌B4和苍白杆菌D7对污泥的VSS去除率次之,分别为48. 6%和47. 5%,比对照组提高16. 8%和14. 2%,均有显著的污泥降解效果。     

A Comparison of AFLP and RAPD Markers for Genetic Diversity and Cultivar Identification in Cotton

AFLP and RAPDmarkers were employed in sixteen diploid cotton (Gossypium sp) cultivars for genetic diversity estimation and cultivar identification. Polymorphism information content (PIC) and percent polymorphism were found to be more for AFLP markers as compared to RAPD markers. Average Jaccard’s genetic similarity index was found to be almost similar using either AFLP or RAPD markers. All the cultivars could be distinguished from one another using AFLP markers and also by the combined RAPD profiles. Cultivar identification indicators like resolving power, marker index and probability of chance identity of two cultivars suggested the usefulness of AFLP markers over the RAPD markers. AFLP and RAPD analyses revealed limited genetic diversity in the studied cultivars. Cluster analysis of both RAPD and AFLP data produced two clusters, one containing cultivars of G. herbaceum and another containing cultivars of G. arboreum species. Highly positive correlation between cophenetic matrices using RAPD and AFLP markers was observed. AFLP markers were found to be more efficient for genetic diversity estimation, polymorphism detection and cultivar identification.     

Chemiluminescent detection of AFLP markers

Nonradioactive amplified fragment-length polymorphism (AFLP) marker detection, a PCR-based, DNA-fingerprinting technique, was achieved by blotting AFLP products after electrophoresis onto a nylon membrane and subsequently hybridizing the blot with an alkaline phosphatase-labeled AFLP probe. Similar AFLP profiles were obtained by both a nonradioactive, chemiluminescent detection technique and by conventional AFLP marker detection using 32P-labeled AFLP primers. The suitability of the method using different gel systems combined with subsequent chemiluminescent detection of AFLP markers is validated by similar dendrograms that were generated using the unweighted pair group method with arithmetic averages (UPGMA). Moreover, chemiluminescent detection of AFLP markers using a universal AFLP nonradioactive probe has been successfully applied on prokaryotes such as Agrobacterium and eukaryotic genomes such as soybean and fungi.     

The inheritance and chromosomal localization of AFLP markers in a non-inbred potato offspring

AFLP^TM is a new technique to generate large numbers of molecular markers for genetic mapping. The method involves the selective amplification of a limited number of DNA restriction fragments out of complex plant genomic DNA digests using PCR. With six primer combinations 264 segregating AFLP amplification products were identified in a diploid backcross population from non-inbred potato parents. The identity of an AFLP marker was specified by the primer combination of the amplification product and its size estimated in bases. The segregating AFLP amplification products were mapped by using a mapping population with 217 already known RFLP, isozyme and morphological trait loci. In general, the AFLP markers were randomly distributed over the genome, although a few clusters were observed. No indications were found that AFLP markers are present in other parts of the genome than those already covered by RFLP markers. Locus specificity of AFLP markers was demonstrated because equally sized amplification products segregating from both parental clones generally mapped to indistinguishable maternal and paternal map positions. Locus specificity of AFLP amplification products will allow to establish the chromosomal identity of linkage groups in future mapping studies.Since AFLP technology is a multi-locus detection system, it was not possible to identify the AFLP alleles which belong to a single AFLP locus. The consequences of a genetic analysis based on single alleles, rather than on loci with two or more alleles on mapping studies using progenies of non-inbred parents are discussed.