Xanthomonas Retroflexus Research Articles

Interspecific interactions facilitate keystone species in a multispecies biofilm that promotes plant growth.

Microorganisms colonizing plant roots co-exist in complex, spatially structured multispecies biofilm communities. However, little is known about microbial interactions and the underlying spatial organization within biofilm communities established on plant roots. Here, a well-established four-species biofilm model (Stenotrophomonas rhizophila, Paenibacillus amylolyticus, Microbacterium oxydans, and Xanthomonas retroflexus, termed as SPMX) was applied to Arabidopsis roots to study the impact of multispecies biofilm on plant growth and the community spatial dynamics on the roots. SPMX co-culture notably promoted root development and plant biomass. Co-cultured SPMX increased root colonization and formed multispecies biofilms, structurally different from those formed by monocultures. By combining 16S rRNA gene amplicon sequencing and fluorescence in situ hybridization with confocal laser scanning microscopy, we found that the composition and spatial organization of the four-species biofilm significantly changed over time. Monoculture P. amylolyticus colonized plant roots poorly, but its population and root colonization were highly enhanced when residing in the four-species biofilm. Exclusion of P. amylolyticus from the community reduced overall biofilm production and root colonization of the three species, resulting in the loss of the plant growth-promoting effects. Combined with spatial analysis, this led to identification of P. amylolyticus as a keystone species. Our findings highlight that weak root colonizers may benefit from mutualistic interactions in complex communities and hereby become important keystone species impacting community spatial organization and function. This work expands the knowledge on spatial organization uncovering interspecific interactions in multispecies biofilm communities on plant roots, beneficial for harnessing microbial mutualism promoting plant growth.

Flagellar interference with plasmid uptake in biofilms: a joint experimental and modeling study.

Plasmid conjugation is a key facilitator of horizontal gene transfer (HGT), and plasmids encoding antibiotic resistance drive the increasing prevalence of antibiotic resistance. In natural, engineered, and clinical environments, bacteria often grow in protective biofilms. Therefore, a better understanding of plasmid transfer in biofilms is needed. Our aim was to investigate plasmid transfer in a biofilm-adapted wrinkly colony mutant of Xanthomonas retroflexus (XRw) with enhanced matrix production and reduced motility. We found that XRw biofilms had an increased uptake of the broad host-range IncP-1ϵ plasmid pKJK5 compared to the wild type (WT). Proteomics revealed fewer flagellar-associated proteins in XRw, suggesting that flagella were responsible for reducing plasmid uptake. This was confirmed by the higher plasmid uptake of non-flagellated fliM mutants of the X. retroflexus wrinkly mutant as well as the wild type. Moreover, testing several flagellar mutants of Pseudomonas putida suggested that the flagellar effect was more general. We identified seven mechanisms with the potential to explain the flagellar effect and simulated them in an individual-based model. Two mechanisms could thus be eliminated (increased distances between cells and increased lag times due to flagella). Another mechanism identified as viable in the modeling was eliminated by further experiments. The possibility of steric hindrance of pilus movement and binding by flagella, reducing the frequency of contact and thus plasmid uptake, proved viable, and the three other viable mechanisms had a reduced probability of plasmid transfer in common. Our findings highlight the important yet complex effects of flagella during bacterial conjugation in biofilms.IMPORTANCEBiofilms are the dominant form of microbial life and bacteria living in biofilms are markedly different from their planktonic counterparts, yet the impact of the biofilm lifestyle on horizontal gene transfer (HGT) is still poorly understood. Horizontal gene transfer by conjugative plasmids is a major driver in bacterial evolution and adaptation, as exemplified by the troubling spread of antibiotic resistance. To either limit or promote plasmid prevalence and dissemination, we need a better understanding of plasmid transfer between bacterial cells, especially in biofilms. Here, we identified a new factor impacting the transfer of plasmids, flagella, which are required for many types of bacterial motility. We show that their absence or altered activity can lead to enhanced plasmid uptake in two bacterial species, Xanthomonas retroflexus and Pseudomonas putida. Moreover, we demonstrate the utility of mathematical modeling to eliminate hypothetical mechanisms.

Metabolic Profiling of Interspecies Interactions During Sessile Bacterial Cultivation Reveals Growth and Sporulation Induction in Paenibacillus amylolyticus in Response to Xanthomonas retroflexus.

The toolbox available for microbiologists to study interspecies interactions is rapidly growing, and with continuously more advanced instruments, we are able to expand our knowledge on establishment and function of microbial communities. However, unravelling molecular interspecies interactions in complex biological systems remains a challenge, and interactions are therefore often studied in simplified communities. Here we perform an in-depth characterization of an observed interspecies interaction between two co-isolated bacteria, Xanthomonas retroflexus and Paenibacillus amylolyticus. Using microsensor measurements for mapping the chemical environment, we show how X. retroflexus promoted an alkalization of its local environment through degradation of amino acids and release of ammonia. When the two species were grown in proximity, the modified local environment induced a morphological change and growth of P. amylolyticus followed by sporulation. 2D spatial metabolomics enabled visualization and mapping of the degradation of oligopeptide structures by X. retroflexus and morphological changes of P. amylolyticus through e.g. the release of membrane-associated metabolites. Proteome analysis and microscopy were used to validate the shift from vegetative growth towards sporulation. In summary, we demonstrate how environmental profiling by combined application of microsensor, microscopy, metabolomics and proteomics approaches can reveal growth and sporulation promoting effects resulting from interspecies interactions.

Emergent bacterial community properties induce enhanced drought tolerance in Arabidopsis

Drought severely restricts plant production and global warming is further increasing drought stress for crops. Much information reveals the ability of individual microbes affecting plant stress tolerance. However, the effects of emergent bacterial community properties on plant drought tolerance remain largely unexplored. Here, we inoculated Arabidopsis plants in vivo with a four-species bacterial consortium (Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans, and Paenibacillus amylolyticus, termed as SPMX), which is able to synergistically produce more biofilm biomass together than the sum of the four single-strain cultures, to investigate its effects on plant performance and rhizo-microbiota during drought. We found that SPMX remarkably improved Arabidopsis survival post 21-day drought whereas no drought-tolerant effect was observed when subjected to the individual strains, revealing emergent properties of the SPMX consortium as the underlying cause of the induced drought tolerance. The enhanced drought tolerance was associated with sustained chlorophyll content and endogenous abscisic acid (ABA) signaling. Furthermore, our data showed that the addition of SPMX helped to stabilize the diversity and structure of root-associated microbiomes, which potentially benefits plant health under drought. These SPMX-induced changes jointly confer an increased drought tolerance to plants. Our work may inform future efforts to engineer the emergent bacterial community properties to improve plant tolerance to drought.

Community-intrinsic properties enhance keratin degradation from bacterial consortia.

Although organic matter may accumulate sometimes (e.g. lignocellulose in peat bog), most natural biodegradation processes are completed until full mineralization. Such transformations are often achieved by the concerted action of communities of interacting microbes, involving different species each performing specific tasks. These interactions can give rise to novel "community-intrinsic" properties, through e.g. activation of so-called "silent genetic pathways" or synergistic interplay between microbial activities and functions. Here we studied the microbial community-based degradation of keratin, a recalcitrant biological material, by four soil isolates, which have previously been shown to display synergistic interactions during biofilm formation; Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans and Paenibacillus amylolyticus. We observed enhanced keratin weight loss in cultures with X. retroflexus, both in dual and four-species co-cultures, as compared to expected keratin degradation by X. retroflexus alone. Additional community intrinsic properties included accelerated keratin degradation rates and increased biofilm formation on keratin particles. Comparison of secretome profiles of X. retroflexus mono-cultures to co-cultures revealed that certain proteases (e.g. serine protease S08) were significantly more abundant in mono-cultures, whereas co-cultures had an increased abundance of proteins related to maintaining the redox environment, e.g. glutathione peroxidase. Hence, one of the mechanisms related to the community intrinsic properties, leading to enhanced degradation from co-cultures, might be related to a switch from sulfitolytic to proteolytic functions between mono- and co-cultures, respectively.

Interspecies interactions reduce selection for a biofilm-optimized variant in a four-species biofilm model.

Multispecies biofilms are structured and spatially defined communities, where interspecies interactions impact assembly and functionality. Here, we compared the spatial organization and growth of bacterial cells in differently composed biofilm communities over time to determine links between interspecies interactions and selection for biofilm phenotypes of individual species. An established model community consisting of Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans and Paenibacillus amylolyticus was used. It was found that interspecies interactions led to varying levels of selection for a new colony phenotype of X. retroflexus, depending on the presence/absence of other species. When M. oxydans was absent, X. retroflexus was not able to establish in the top layers of the biofilm, which led to selection for a hyper-matrix forming phenotype of X. retroflexus that successfully established in the biofilm top layers. No such phenotypic X. retroflexus variants were identified in the presence of M. oxydans. These findings indicate that interspecies interactions may lead to favourable localization of individual species in a multispecies biofilm and thereby reduce selection for competitive phenotypes.

Interspecies interactions reduce selection for a biofilm optimized variant in a four-species biofilm model.

Multispecies biofilms are structured and spatially defined communities, where interspecies interactions impact assembly and functionality. Here we compared the spatial organization and growth of bacterial cells in differently composed biofilm communities over time to determine links between interspecies interactions and selection for biofilm phenotypes of individual species. An established model community consisting of Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans and Paenibacillus amylolyticus was used. It was found that interspecies interactions led to varying levels of selection for a new colony phenotype of X. retroflexus, depending on the presence/absence of other species. When M. oxydans was absent, X. retroflexus was not able to establish in the top layers of the biofilm, which led to selection for a hyper-matrix forming phenotype of X. retroflexus that successfully established in the biofilm top layers. No such phenotypic X. retroflexus variants were identified in the presence of M. oxydans. These findings indicate that interspecies interactions may lead to favourable localization of individual species in a multispecies biofilm and thereby reduce selection for competitive phenotypes. This article is protected by copyright. All rights reserved.

Priority of Early Colonizers but No Effect on Cohabitants in a Synergistic Biofilm Community.

The arrival order of different species to a habitat can strongly impact community assembly and succession dynamics, thus influencing functionality. In this study, we asked how prior colonization of one community member would influence the assembly of a synergistic multispecies biofilm community grown in vitro. We expected that the prior arrival would confer an advantage, in particular for good biofilm formers. Yet, we did not know if the cohabitants would be impaired or benefit from the pre-colonization of one member, depending on its ability to form biofilm. We used a consortium consisting of four soil bacteria; Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans and Paenibacillus amylolyticus. This consortium has been shown to act synergistically when grown together, thus increasing biofilm production. The results showed that the two good biofilm formers gained a fitness advantage (increase in abundance) when allowed prior colonization on an abiotic surface before the arrival of their cohabitants. Interestingly, the significantly higher number of the pre-colonized biofilm formers did not affect the resulting composition in the subsequent biofilm after 24 h.

Enhanced bacterial mutualism through an evolved biofilm phenotype.

Microbial communities primarily consist of multiple species that affect one another's fitness both directly and indirectly. This study showed that the cocultivation of Paenibacillus amylolyticus and Xanthomonas retroflexus exhibited facultative mutualistic interactions in a static environment, during the course of which a new adapted phenotypic variant of X. retroflexus appeared. Although the emergence of this variant was not directly linked to the presence of P. amylolyticus, its establishment in the coculture enhanced the productivity of both species due to mutations that stimulated biofilm formation. The mutations were detected in genes encoding a diguanylate cyclase predicted to synthesise cyclic-di-GMP. Examinations of the biofilm formed in cocultures of P. amylolyticus and the new variant of X. retroflexus revealed a distinct spatial organisation: P. amylolyticus only resided in biofilms in association with X. retroflexus and occupied the outer layers. The X. retroflexus variant therefore facilitated increased P. amylolyticus growth as it produced more biofilm biomass. The increase in X. retroflexus biomass was thus not at the expense of P. amylolyticus, demonstrating that interspecies interactions can shape diversification in a mutualistic coculture and reinforce these interactions, ultimately resulting in enhanced communal performance.

Micro-scale intermixing: a requisite for stable and synergistic co-establishment in a four-species biofilm

Microorganisms frequently coexist in complex multispecies communities, where they distribute non-randomly, reflective of the social interactions that occur. It is therefore important to understand how social interactions and local spatial organization influences multispecies biofilm succession. Here the localization of species pairs was analyzed in three dimensions in a reproducible four-species biofilm model, to study the impact of spatial positioning of individual species on the temporal development of the community. We found, that as the biofilms developed, species pairs exhibited distinct intermixing patterns unique to the four-member biofilms. Higher biomass and more intermixing were found in four-species biofilms compared to biofilms with fewer species. Intriguingly, in local regions within the four member biofilms where Microbacterium oxydans was scant, both biomass and intermixing of all species were lowered, compared to regions where M. oxydans was present at typical densities. Our data suggest that Xanthomonas retroflexus and M. oxydans, both low abundant biofilm-members, intermixed continuously during the development of the four-species biofilm, hereby facilitating their own establishment. In turn, this seems to have promoted distinct spatial organization of Stenotrophomonas rhizophila and Paenibacillus amylolyticus enabling enhanced growth of all four species. Here local intermixing of bacteria advanced the temporal development of a multi-species biofilm.

Synergistic Interactions within a Multispecies Biofilm Enhance Individual Species Protection against Grazing by a Pelagic Protozoan.

Biofilm formation has been shown to confer protection against grazing, but little information is available on the effect of grazing on biofilm formation and protection in multispecies consortia. With most biofilms in nature being composed of multiple bacterial species, the interactions and dynamics of a multispecies bacterial biofilm subject to grazing by a pelagic protozoan predator were investigated. To this end, a mono and multispecies biofilms of four bacterial soil isolates, namely Xanthomonas retroflexus, Stenotrophomonas rhizophila, Microbacterium oxydans and Paenibacillus amylolyticus, were constructed and subjected to grazing by the ciliate Tetrahymena pyriformis. In monocultures, grazing strongly reduced planktonic cell numbers in P. amylolyticus and S. rhizophila and also X. retroflexus. At the same time, cell numbers in the underlying biofilms increased in S. rhizophila and X. retroflexus, but not in P. amylolyticus. This may be due to the fact that while grazing enhanced biofilm formation in the former two species, no biofilm was formed by P. amylolyticus in monoculture, either with or without grazing. In four-species biofilms, biofilm formation was higher than in the best monoculture, a strong biodiversity effect that was even more pronounced in the presence of grazing. While cell numbers of X. retroflexus, S. rhizophila, and P. amylolyticus in the planktonic fraction were greatly reduced in the presence of grazers, cell numbers of all three species strongly increased in the biofilm. Our results show that synergistic interactions between the four-species were important to induce biofilm formation, and suggest that bacterial members that produce more biofilm when exposed to the grazer not only protect themselves but also supported other members which are sensitive to grazing, thereby providing a “shared grazing protection” within the four-species biofilm model. Hence, complex interactions shape the dynamics of the biofilm and enhance overall community fitness under stressful conditions such as grazing. These emerging inter- and intra-species interactions could play a vital role in biofilm dynamics in natural environments like soil or aquatic systems.

A meta-proteomics approach to study the interspecies interactions affecting microbial biofilm development in a model community

Microbial biofilms are omnipresent in nature and relevant to a broad spectrum of industries ranging from bioremediation and food production to biomedical applications. To date little is understood about how multi-species biofilm communities develop and function on a molecular level, due to the complexity of these biological systems. Here we apply a meta-proteomics approach to investigate the mechanisms influencing biofilm formation in a model consortium of four bacterial soil isolates; Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans and Paenibacillus amylolyticus. Protein abundances in community and single species biofilms were compared to describe occurring inter-species interactions and the resulting changes in active metabolic pathways. To obtain full taxonomic resolution between closely related species and empower correct protein quantification, we developed a novel pipeline for generating reduced reference proteomes for spectral database searches. Meta-proteomics profiling indicated that community development is dependent on cooperative interactions between community members facilitating cross-feeding on specific amino acids. Opposite regulation patterns of fermentation and nitrogen pathways in Paenibacillus amylolyticus and Xanthomonas retroflexus may, however, indicate that competition for limited resources also affects community development. Overall our results demonstrate the multitude of pathways involved in biofilm formation in mixed communities.

Low-abundant species facilitates specific spatial organization that promotes multispecies biofilm formation.

Microorganisms frequently co-exist in matrix-embedded multispecies biofilms. Within biofilms, interspecies interactions influence the spatial organization of member species, which likely play an important role in shaping the development, structure and function of these communities. Here, a reproducible four-species biofilm, composed of Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans and Paenibacillus amylolyticus, was established to study the importance of individual species spatial organization during multispecies biofilm development. We found that the growth of species that are poor biofilm formers, M. oxydans and P. amylolyticus, were highly enhanced when residing in the four-species biofilm. Interestingly, the presence of the low-abundant M. oxydans (0.5% of biomass volume) was observed to trigger changes in the composition of the four-species community. The other three species were crucially needed for the successful inclusion of M. oxydans in the four-species biofilm, where X. retroflexus was consistently positioned in the top layer of the mature four-species biofilm. These findings suggest that low abundance key species can significantly impact the spatial organization and hereby stabilize the function and composition of complex microbiomes.

Distinct gene expression profile of Xanthomonas retroflexus engaged in synergistic multispecies biofilm formation.

It is well known that bacteria often exist in naturally formed multispecies biofilms. Within these biofilms, interspecies interactions seem to have an important role in ecological processes. Little is known about the effects of interspecies interactions on gene expression in these multispecies biofilms. This study presents a comparative gene expression analysis of the Xanthomonas retroflexus transcriptome when grown in a single-species biofilm and in dual- and four-species consortia with Stenotrophomonas rhizophila, Microbacterium oxydans and Paenibacillus amylolyticus. The results revealed complex interdependent interaction patterns in the multispecies biofilms. Many of the regulated functions are related to interactions with the external environment and suggest a high phenotypic plasticity in response to coexistence with other species. Furthermore, the changed expression of genes involved in aromatic and branched-chain amino acid biosynthesis suggests nutrient cross feeding as a contributing factor for the observed synergistic biofilm production when these four species coexists in a biofilm.

Zinc Solubilizing Bacterial Isolates from the Agricultural Fields of Coimbatore, Tamil Nadu, India

Zinc plays a pivotal role in physiological and biochemical functions of the plant. Both quantitative and qualitative yield of the plant is strongly dependent on this micronutrient. Supplementation of zinc in the form of synthetic fertilizer is proved to be inappropriate due to its unavailability to plants. This crisis can be prevented by the identification of rhizospheric micro- organisms which has the potential to transform various unavailable forms of the metal to available forms. In the present study about thirty five zinc solubilizing bacteria were isolated from eight different agricultural fields (banana, chilli, field bean, ground nut, maize, sugarcane, sorghum and tomato) in and around Coimbatore district of Tamil Nadu. Five isolates were selected as best strains based on their solubilization efficacy in the qualitative estimation. The selected five isolates were identified using 16S rRNA as Stenotrophomonas maltophilia (ZSB-1), Mycobacterium brisbanense (ZSB-10), Enterobacter aerogenes (ZSB-13), Pseudomonas aeruginosa (ZSB-22) and Xanthomonas retroflexus (ZSB-23). These strains were subjected to further studies such as quantitative estimation influence of the isolates on the pH of the medium and production of gluconic acid as well as IAA. Of the five bacterial isolates, Pseudomonas aeruginosa showed maximum solubilization of zinc in the broth and also maximum decrease in the pH from 7 to 3.3 and recorded highest IAA production. HPLC analysis of gluconic acid production by the selected isolates indicated their potential to solubilize zinc.

High prevalence of biofilm synergy among bacterial soil isolates in cocultures indicates bacterial interspecific cooperation.

Biofilms that form on roots, litter and soil particles typically contain multiple bacterial species. Currently, little is known about multispecies biofilm interactions and few studies have been based on environmental isolates. Here, the prevalence of synergistic effects in biofilm formation among seven different soil isolates, cocultured in combinations of four species, was investigated. We observed greater biofilm biomass production in 63% of the four-species culture combinations tested than in biofilm formed by single-species cultures, demonstrating a high prevalence of synergism in multispecies biofilm formation. One four-species consortium, composed of Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans and Paenibacillus amylolyticus, exhibited strong synergy in biofilm formation and was selected for further study. Of the four strains, X. retroflexus was the only one capable of forming abundant biofilm in isolation, under the in vitro conditions investigated. In accordance, strain-specific quantitative PCR revealed that X. retroflexus was predominant within the four-species consortium (>97% of total biofilm cell number). Despite low relative abundance of all the remaining strains, all were indispensable for the strong synergistic effect to occur within the four-species biofilm. Moreover, absolute individual strain cell numbers were significantly enhanced when compared with those of single-species biofilms, indicating that all the individual strains benefit from inclusion in the multispecies community. Our results show a high prevalence of synergy in biofilm formation in multispecies consortia isolated from a natural bacterial habitat and suggest that interspecific cooperation occurs.

High-Throughput Screening of Multispecies Biofilm Formation and Quantitative PCR-Based Assessment of Individual Species Proportions, Useful for Exploring Interspecific Bacterial Interactions

Multispecies biofilms are predominant in almost all natural environments, where myriads of resident microorganisms interact with each other in both synergistic and antagonistic manners. The interspecies interactions among different bacteria are, despite the ubiquity of these communities, still poorly understood. Here, we report a rapid, reproducible and sensitive approach for quantitative screening of biofilm formation by bacteria when cultivated as mono- and multispecies biofilms, based on the Nunc-TSP lid system and crystal violet staining. The relative proportion of the individual species in a four-species biofilm was assessed using quantitative PCR based on SYBR Green I fluorescence with specific primers. The results indicated strong synergistic interactions in a four-species biofilm model community with a more than 3-fold increase in biofilm formation and demonstrated the strong dominance of two strains, Xanthomonas retroflexus and Paenibacillus amylolyticus. The developed approach can be used as a standard procedure for evaluating interspecies interactions in defined microbial communities. This will be of significant value in the quantitative study of the microbial composition of multispecies biofilms both in natural environments and infectious diseases to increase our understanding of the mechanisms that underlie cooperation, competition and fitness of individual species in mixed-species biofilms.

Adhesion to brown trout skin mucus, antagonism against cyst adhesion and pathogenicity to rainbow trout of some inhibitory bacteria against Saprolegnia parasitica

Biological control of saprolegniosis with bacteria might be an alternative to the use of chemical compounds. Among criteria for the selection of such bacteria are their absence of pathogenicity to fish and their ability to prevent adhesion of the pathogen to the skin mucus. The pathogenicity to rainbow trout of 21 bacterial isolates with in vitro inhibitory activity against Saprolegnia parasitica was studied. Fifteen of the isolates, identified as Aeromonas sobria, Pantoea agglomerans, Pseudomonas fluorescens, Serratia fonticola, Xanthomonas retroflexus and Yersinia kristensenii, were non-pathogenic when injected into rainbow trout. Their capacity to adhere to the skin mucus of male and female brown trout and to reduce the adhesion of S. parasitica cysts under exclusion, competition and displacement conditions was tested. The 15 bacterial isolates showed a low adhesion rate, ranging between 1.7% (for an A. sobria isolate) and 15.3% (a P. fluorescens isolate). This adhesion was greater in the case of mucus from male brown trout than from females. Similarities in the adhesion to male mucus and other substrates and correlation to that observed to polystyrene suggest that adhesion to skin mucus does not depend on the substrate. A high percentage (88.9%) of the S. parasitica cysts adhered to the skin mucus of male brown trout. Almost all of the bacteria reduced this adhesion ratio significantly under exclusion and competition conditions. However, only half of the isolates displaced cysts from skin mucus, and more bacterial cells were necessary for this effect. A novel method to study the adhesion of S. parasitica cysts to skin mucus of trout and their interactions with inhibitory bacteria is described.

Environmental significance of O‐demethylation of chloroanisoles by soil bacterial isolates as a mechanism that improves the overall biodegradation of chlorophenols

The biodegradation rate of chlorophenols in the environment seems to be limited by a competitive mechanism of O-methylation which produces chloroanisoles with a high potential of being bioconcentrated in living organisms. In this work we report for the first time the isolation of three soil bacterial strains able to efficiently degrade 2,4,6-trichloroanisole (2,4,6-TCA). These strains were identified as Xanthomonas retroflexus INBB4, Pseudomonas putida INBP1 and Acinetobacter radioresistens INBS1. In these isolates 2,4,6-TCA was efficiently metabolized in a minimal medium containing methanol and 2,4,6-TCA as the only carbon sources, with a concomitant release of 3 mol of chloride ion from 1 mol of 2,4,6-TCA, indicating complete dehalogenation of 2,4,6-TCA. 2,4,6-trichlorophenol (2,4,6-TCP) was identified as a degradative intermediate, indicating that 2,4,6-TCA underwent O-demethylation as the first step in the biodegradation process. 2,4,6-TCP was further transformed into 2,6-dichloro-para-hydroquinone (2,6-DCHQ) and subsequently mineralized. The degradation of chloroanisoles could improve the overall biodegradation of chlorophenols in the environment, because those chlorophenols previously biomethylated might also be later biodegraded. Xanthomonas retroflexus INBB4 has two O-demethylation systems: one is an oxygenase-type demethylase, and the other is a tetrahydrofolate (THF)-dependent O-demethylase. On the contrary O-demethylation of 2,4,6-TCA in P. putida INBP1 is just catalysed by an oxygenase-type NADH/NADPH-dependent O-demethylase, whereas in A. radioresistens INBS1 a THF-dependent O-demethylase activity was detected.

Physiological effect of the toxin from Xanthomonas retroflexus on redroot pigweed (Amaranthus retroflexus)

A new toxin from Xanthomonas retroflexus could cause a series of physiological responses on seedlings of redroot pigweed. The experimental results revealed that respiratory ratio first increased 106% after treatment with the CFS (concentrated centrifugal fermentation supernatants) of X. retroflexus for 2 h, and decreased after treatment for 3 h; peroxidase (POD) activity increased 30% and 50% after treatment with CFS for 1 h and 2 h, respectively; Malon dialdehyde (MDA), soluble protein and mitotic indices appeared not to be affected by the toxin. Ultrastructure observation indicated that the thylakoids of chloroplast and cristae of mitochondria swelled, when the leaves were placed in the toxin for 2 h. After treatment with the phototoxin for 3 h, the cell membrane was disrupted, the chloroplasts disintegrated and the mitochondria vesiculated.