Genetic Diversity among 3-Chloroaniline- and Aniline-Degrading Strains of the Comamonadaceae

We examined the diversity of the plasmids and of the gene tdnQ, involved in the oxidative deamination of aniline, in five bacterial strains that are able to metabolize both aniline and 3-chloroaniline (3-CA). Three strains have been described and identified previously, i.e., Comamonas testosteroni I2 and Delftia acidovorans CA28 and BN3.1. Strains LME1 and B8c were isolated in this study from linuron-treated soil and from a wastewater treatment plant, respectively, and were both identified as D. acidovorans. Both Delftia and Comamonas belong to the family Comamonadaceae. All five strains possess a large plasmid of ca. 100 kb, but the plasmids from only four strains could be transferred to a recipient strain by selection on aniline or 3-CA as a sole source of carbon and/or nitrogen. Plasmid transfer experiments and Southern hybridization revealed that the plasmid of strain I2 was responsible for total aniline but not 3-CA degradation, while the plasmids of strains LME1 and B8c were responsible only for the oxidative deamination of aniline. Several transconjugant clones that had received the plasmid from strain CA28 showed different degradative capacities: all transconjugants could use aniline as a nitrogen source, while only some of the transconjugants could deaminate 3-CA. For all four plasmids, the IS1071 insertion sequence of Tn5271 was found to be located on a 1.4-kb restriction fragment, which also hybridized with the tdnQ probe. This result suggests the involvement of this insertion sequence element in the dissemination of aniline degradation genes in the environment. By use of specific primers for the tdnQ gene from Pseudomonas putida UCC22, the diversity of the PCR-amplified fragments in the five strains was examined by denaturing gradient gel electrophoresis (DGGE). With DGGE, three different clusters of the tdnQ fragment could be distinguished. Sequencing data showed that the tdnQ sequences of I2, LME1, B8c, and CA28 were very closely related, while the tdnQ sequences of BN3.1 and P. putida UCC22 were only about 83% identical to the other sequences. Northern hybridization revealed that the tdnQ gene is transcribed only in the presence of aniline and not when only 3-CA is present.     

Genes involved in aniline degradation by Delftia acidovorans strain 7N and its distribution in the natural environment

Aniline-degraders were isolated from activated sludge and environmental samples and classified into eight phylogenetic groups. Seven groups were classified into Gram-negative bacteria, such as Acidovorax sp., Acinetobacter sp., Delftia sp., Comamonas sp., and Pseudomonas sp., suggesting the possible dominance of Gram-negative aniline-degraders in the environment. Aniline degradative genes were cloned from D. acidovorans strain 7N, and the nucleotide sequence of the 8,039-bp fragment containing eight open reading frames was determined. Their deduced amino acid sequences showed homologies to glutamine synthetase (GS)-like protein, glutamine amidotransferase (GA)-like protein, large and small subunits of aniline dioxygenase, reductase, LysR-type regulator, small ferredoxin-like protein, and catechol 2,3-dioxygenase, suggesting a high similarity of this gene cluster to those in P. putida strain UCC22 and Acinetobacter sp. strain YAA. Polymerase chain reaction (PCR) and sequencing analyses of GS-like protein gene segments of other Gram-negative bacteria suggested that Gram-negative bacteria have aniline degradative gene that can be divided into two distinctive groups.     

Nucleotide sequences and regulational analysis of genes involved in conversion of aniline to catechol in Pseudomonas putida UCC22(pTDN1).

A 9,233-bp HindIII fragment of the aromatic amine catabolic plasmid pTDN1, isolated from a derivative of Pseudomonas putida mt-2 (UCC22), confers the ability to degrade aniline on P. putida KT2442. The fragment encodes six open reading frames which are arranged in the same direction. Their 5' upstream region is part of the direct-repeat sequence of pTDN1. Nucleotide sequence of 1.8 kb of the repeat sequence revealed only a single base pair change compared to the known sequence of IS1071 which is involved in the transposition of the chlorobenzoate genes (C. Nakatsu, J. Ng, R. Singh, N. Straus, and C. Wyndham, Proc. Natl. Acad. Sci. USA 88:8312-8316, 1991). Four open reading frames encode proteins with considerable homology to proteins found in other aromatic-compound degradation pathways. On the basis of sequence similarity, these genes are proposed to encode the large and small subunits of aniline oxygenase (tdnA1 and tdnA2, respectively), a reductase (tdnB), and a LysR-type regulatory gene (tdnR). The putative large subunit has a conserved [2Fe-2S]R Rieske-type ligand center. Two genes, tdnQ and tdnT, which may be involved in amino group transfer, are localized upstream of the putative oxygenase genes. The tdnQ gene product shares about 30% similarity with glutamine synthetases; however, a pUC-based plasmid carrying tdnQ did not support the growth of an Escherichia coli glnA strain in the absence of glutamine. TdnT possesses domains that are conserved among amidotransferases. The tdnQ, tdnA1, tdnA2, tdnB, and tdnR genes are essential for the conversion of aniline to catechol.     

Conjugal transfer of plasmid pNB2 to activated sludge bacteria leads to 3-chloroaniline degradation in enrichment cultures

AIMS: The involvement of the aniline-degradative plasmid pNB2 in degradation of 3-chloroaniline (3-CA) was investigated. METHODS AND RESULTS: Plate matings of a Pseudomonas putida strain containing pNB2 with a mixed bacterial culture derived from activated sludge was carried out. After inoculation of the mating mixtures into batch cultures containing 3-CA, degradation of the compound was observed. A total of five different transconjugant strains could be isolated from one of the batch cultures and two of them were able to degrade 3-CA. These two isolates were identified as Comamonas testosteroni by partial 16S rDNA sequencing. CONCLUSIONS: It can be assumed that pNB2 carries a part of the genes involved in the catabolism of 3-CA, but that completion of the pathway must be provided by chromosomal genes in the host strain. SIGNIFICANCE AND IMPACT OF THE STUDY: pNB2 is a candidate plasmid which can be used in plasmid-mediated bioaugmentation of wastewater bacteria involved in degradation of chlorinated anilines.     

Plasmid participation in the degradation of alpha-methylstyrene

Pseudomonas acidovorans 9 transforming alpha-methylstyrene into acetophenone contains four types of plasmid DNA with molecular masses of 130, 110, 36 and 54 MD. The loss of the "growth on alpha-methylstyrene" property by this strain correlates with the absence of plasmids with the molecular masses of 130 and 110 MD from the cells. All the types of plasmid DNA are found in transconjugants growing on alpha-methylstyrene and produced by crossing the parent P. acidovorans strain with the plasmidless variant of this strain incapable of alpha-methylstyrene transformation. Apparently, plasmids with the molecular masses of 130 and 110 MD participate in the genetic control of alpha-methylstyrene transformation into acetophenone by P. acidovorans 9.     

Plasmid-mediated bioaugmentation of activated sludge bacteria in a sequencing batch moving bed reactor using pNB2

AIMS: The applicability of plasmid pNB2 for bioaugmentation of bacteria in model wastewater treatment reactors receiving 3-chloroaniline (3-CA) was investigated. METHODS AND RESULTS: A setup of three biofilm reactors was studied, all initially inoculated with bacteria from activated sludge. Reactor PB received a Pseudomonas putida pNB2 donor strain not able to degrade 3-CA. Positive control reactor P received a 3-CA degrading Comamonas testosteroni pNB2-transconjugant. The negative control reactor N remained unchanged. Reactor P showed 3-CA degradation from the beginning of the experiment whereas in reactor PB, degradation started after an initial lag period. No degradation was observed in reactor N. PCR analysis showed that the P. putida donor abundance dropped in reactor PB, whereas the plasmid abundance did not, indicating transfer to other bacteria. A number of different 3-CA degrading C. testosteroni strains carrying pNB2 could be isolated from reactor PB. CONCLUSIONS: A successful plasmid-mediated bioaugmentation was achieved with C. testosteroni being the dominant 3-CA degrading pNB2 transconjugant species active in reactor PB. SIGNIFICANCE AND IMPACT OF THE STUDY: The study underlines the potential of gene transfer to contribute to establishment and spread of genetic information in general, particularly emphasizing the spread of xenobiotic degrading potential by dissemination of catabolic genes.     

Characterization of strain HY99, a novel microorganism capable of aerobic and anaerobic degradation of aniline

We have characterized a novel microorganism, strain HY99, which is capable of aerobic and anaerobic degradation of aniline. Strain HY99 was found to aerobically metabolize aniline via catechol and 2-hydroxymuconic semialdehyde intermediates, and to transform aniline via p-aminobenzoate in anaerobic environments. Physiological and biochemical tests revealed that strain HY99 was most similar to Delftia acidovorans, but unlike D. acidovorans, strain HY99 was able to metabolize aniline under anaerobic conditions linked with nitrate reduction. Phylogenetic analysis based on 16S rDNA sequencing also revealed that strain HY99 was closely related to D. acidovorans, with 96% overall similarity.     

Degradation of aniline and monochlorinated anilines by soil-born Pseudomonas acidovorans strains

Four bacterial strains (CA26, CA28, CA37, and CA45), which all were able to use aniline, 3-chloroaniline (3-CA), and 4-chloroaniline (4-CA) as sole sources of carbon, nitrogen and energy, were isolated after enrichment in aerated soil columns and identified as Pseudomonas acidovorans strains. In addition strains CA26 and CA45 were able to degrade 2-chloroaniline (2-CA) at very low rates. At 25°C strain CA28 was grown on aniline and 3-CA with generation times of 3.0 and 7.7 h, respectively, and exhibited complete mineralization of these substrates in degradation rates of 2.25 mmol aniline and 1.63 mmol 3-CA g^-1 of biomass per hour, respectively. Degradation of 4-CA occurred at 1.54 mmol 4-CA g^-1 of biomass per hour and a generation time of 18.7 h but, in contrast, was not complete due to formation of minor amounts of chlorohydroxymuconic semialdehyde, a meta-cleavage product of 4-chlorocatechol. The initial attack on the substrate, the formation of corresponding chlorocatechols from 3-CA and 4-CA, was found to be the rate-limiting degradation step. Evidence for two different aniline-oxygenase systems in strain CA28 with distinct activity pattern on chlorinated and nonsubstituted anilines was demonstrated by oxygen uptake rate experiments with aniline and chloroaniline pregrown cells. Further degradation was shown to be initialized by catechol dioxygenases.Non-standard abbreviations CA chloroaniline - DCA dichloroaniline - ECM enrichment and cultivation medium - CFU colony forming unit     

Detection and characterization of conjugative degradative plasmids in xenobiotic-degrading Sphingomonas strains

A systematic survey for the presence of plasmids in 17 different xenobiotic-degrading Sphingomonas strains was performed. In almost all analyzed strains, two to five plasmids with sizes of about 50 to 500 kb were detected by using pulsed-field gel electrophoresis. A comparison of plasmid preparations untreated or treated with S1 nuclease suggested that, in general, Sphingomonas plasmids are circular. Hybridization experiments with labeled gene probes suggested that large plasmids are involved in the degradation of dibenzo-p-dioxin, dibenzofuran, and naphthalenesulfonates in S. wittichii RW1, Sphingomonas sp. HH69, and S. xenophaga BN6, respectively. The plasmids which are responsible for the degradation of naphthalene, biphenyl, and toluene by S. aromaticivorans F199 (pNL1) and of naphthalenesulfonates by S. xenophaga BN6 (pBN6) were site-specifically labeled with a kanamycin resistance cassette. The conjugative transfer of these labeled plasmids was attempted with various bacterial strains as putative recipient strains. Thus, a conjugative transfer of plasmid pBN6 from S. xenophaga BN6 to a cured mutant of strain BN6 and to Sphingomonas sp. SS3 was observed. The conjugation experiments with plasmid pNL1 suggested a broader host range of this plasmid, because it was transferred without any obvious structural changes to S. yanoikuyae B1, Sphingomonas sp. SS3, and S. herbicidovorans. In contrast, major plasmid rearrangements were observed in the transconjugants after the transfer of plasmid pNL1 to Sphingomonas sp. HH69 and of pBN6 to Sphingomonas sp. SS3. No indications for the transfer of a Sphingomonas plasmid to bacteria outside of the Sphingomonadaceae were obtained.     

Plasmid donor affects host range of promiscuous IncP-1beta plasmid pB10 in an activated-sludge microbial community

Horizontal transfer of multiresistance plasmids in the environment contributes to the growing problem of drug-resistant pathogens. Even though the plasmid host cell is the primary environment in which the plasmid functions, possible effects of the plasmid donor on the range of bacteria to which plasmids spread in microbial communities have not been investigated. In this study we show that the host range of a broad-host-range plasmid within an activated-sludge microbial community was influenced by the donor strain and that various mating conditions and isolation strategies increased the diversity of transconjugants detected. To detect transconjugants, the plasmid pB10 was marked with lacp-rfp, while rfp expression was repressed in the donors by chromosomal lacI(q). The phylogeny of 306 transconjugants obtained was determined by analysis of partial 16S rRNA gene sequences. The transconjugants belonged to 15 genera of the alpha- and gamma-Proteobacteria. The phylogenetic diversity of transconjugants obtained in separate matings with donors Pseudomonas putida SM1443, Ralstonia eutropha JMP228, and Sinorhizobium meliloti RM1021 was significantly different. For example, the transconjugants obtained after matings in sludge with S. meliloti RM1021 included eight genera that were not represented among the transconjugants obtained with the other two donors. Our results indicate that the spectrum of hosts to which a promiscuous plasmid transfers in a microbial community can be strongly influenced by the donor from which it transfers.     

IncP-7 naphthalene-degradative plasmids from Pseudomonas putida

Abstract Seven naphthalene-degrading and two naphthalene and camphor-degrading Pseudomonas putida strains were isolated from marine sediments. Most of them carried two plasmids, of molecular size 60 and 200 kb. The naphthalene and salicylate metabolism determinants were transferred to a P. putida strain by conjugation, and the transconjugants acquired either both plasmids or only the 200-kb one. These plasmids appear to belong to the IncP-7 group and encode for catabolism of naphthalene and salicylate, but not camphor.     

Manure Enhances Plasmid Mobilization and Survival of Pseudomonas putida Introduced into Field Soil

We report a field study on plasmid mobilization in an agricultural soil. The influence of pig manure on the mobilization of the IncQ plasmid pIE723 by indigenous plasmids or by the IncP(alpha) plasmid pGP527 into the recipient Pseudomonas putida UWC1 (Rif(supr) Nal(supr)) was studied in field soil. Six plots were prepared in duplicate, three of which were treated with manure prior to inoculation of the donor and recipient strains. As a donor strain, either Escherichia coli J53(pIE723) or E. coli 600(pIE723, pGP527) was used. Putative transconjugants obtained on a selective medium were confirmed by DNA hybridization and PCR. Plasmid mobilization by indigenous mobilizing plasmids was observed on two occasions in manured soil. Manuring of soil significantly enhanced the frequency of pIE723 mobilization by pGP527, since mobilization frequencies into P. putida UWC1 were at least 10-fold higher in manured soil than in nonmanured soil. Enhanced numbers of P. putida UWC1 transconjugant and recipient colonies could be observed in manured soil throughout the 79-day field test. Transfer of pIE723 or pG527 into indigenous soil or rhizosphere bacteria could not be detected when indigenous bacteria isolated by selective cultivation were screened for the presence of these plasmids by DNA hybridization. Furthermore, the presence of IncN-, IncP-, or IncQ-specific sequences was confirmed in total community DNA extracted directly from the manured or nonmanured soil by PCR. IncW plasmids were detectable only in manured soil, indicating entry of these plasmids into soil via manure.     

Escherichia coli K317, formerly used to define colicin group E2, produces colicin E7, is immune to colicin E2, and carries a bacteriophage-restricting conjugative plasmid.

Escherichia coli strain CL137, a K-12 derivative made E colicinogenic by contact with Fredericq's strain K317, was unaffected by colicin E2-P9, but K-12 carrying ColE2-P9 was sensitive to the E colicin made by strains CL137 and K317. This colicin we named E7-K317 because by the test of colicinogenic immunity it differed from colicins E1-K30, E2-P9, and E3-CA38 and from recently recognized colicins termed E4Horak, E5, and E6. Strain K317 as conjugational donor transmitted E7 colicinogeny; about half the E7-colicinogenic transconjugants were immune to colicin E2-P9. A spontaneous variant of CL137 retained E7 colicinogeny but was sensitive to E2 colicins. We attribute the E2 immunity of strain CL137 and some E7-coliconogeic transconjugants to a "colicin-immunity plasmid," ColE2imm-K317, from strain K317. Tra+ E7-colicinogenic transconjugants restricted phage BF23 in the same way as strains carrying ColIb-P9. We attribute Tra+ and restricting ability to a plasmid, pRES-K317, acquired from strain K317, and related to the ColI plasmids.     

Transposition of a beta-lactamase locus from RP1 into Pseudomonas putida degradative plasmids.

The beta-lactamase gene from the RP1 plasmid transposes into at least two Pseudomonas putida degradative plasmids. Donor strains that carry RP1 (bla+ tet+ aphA+) and a degradative plasmid yield transconjugants that have only the bla+ marker of RP1. This occurs in up to 80% of all bla+ transconjugants. Segregation of the bla+ marker requires the presence of a degradative plasmid in the donor and is only observed in transconjugants that have received degradative markers. The bla+ tet aphA transconjugants show 100% linkage of bla+ to degradative markers in conjugation,transduction, and transformation crosses. A transduction cross of an (RP1), (SAL) donor shows that 8% of all SAL plasmids also carry the transposed bla+ marker. Tn401 is the name we assign to the bla+ transposon from RP1 observed in Pseudomonas. Its identity with the RP1 bla+ transposon observed in Escherichia coli is not known. In four cases, Tn401 has inserted into the camphor genes of the CAM-OCT plasmid.     

Synergistic degradation of linuron by a bacterial consortium and isolation of a single linuron-degrading variovorax strain

The bacterial community composition of a linuron-degrading enrichment culture and the role of the individual strains in linuron degradation have been determined by a combination of methods, such as denaturing gradient gel electrophoresis of the total 16S rRNA gene pool, isolation and identification of strains, and biodegradation assays. Three strains, Variovorax sp. strain WDL1, Delftia acidovorans WDL34, and Pseudomonas sp. strain WDL5, were isolated directly from the linuron-degrading culture. In addition, subculture of this enrichment culture on potential intermediates in the degradation pathway of linuron (i.e., N,O-dimethylhydroxylamine and 3-chloroaniline) resulted in the isolation of, respectively, Hyphomicrobium sulfonivorans WDL6 and Comamonas testosteroni WDL7. Of these five strains, only Variovorax sp. strain WDL1 was able to use linuron as the sole source of C, N, and energy. WDL1 first converted linuron to 3,4-dichloroaniline (3,4-DCA), which transiently accumulated in the medium but was subsequently degraded. To the best of our knowledge, this is the first report of a strain that degrades linuron further than the aromatic intermediates. Interestingly, the rate of linuron degradation by strain WDL1 was lower than that for the consortium, but was clearly increased when WDL1 was coinoculated with each of the other four strains. D. acidovorans WDL34 and C. testosteroni WDL7 were found to be responsible for degradation of the intermediate 3,4-DCA, and H. sulfonivorans WDL6 was the only strain able to degrade N,O-dimethylhydroxylamine. The role of Pseudomonas sp. strain WDL5 needs to be further elucidated. The degradation of linuron can thus be performed by a single isolate, Variovorax sp. strain WDL1, but is stimulated by a synergistic interaction with the other strains isolated from the same linuron-degrading culture.     

New plasmids of herbicide 2,4-dichlorophenoxyacetic acid biodegradation

Three herbicide 2,4-D metabolizing bacterial strains were isolated from three independent soil samples of Estonia. The strains, although belonging to various species, contain 2,4-D degradative plasmids with identical restriction patterns. pEST4001 is a 78 kb conjugative plasmid. All Pseudomonas putida PaW340 2,4-D+ transconjugants obtained a 70 kb plasmid pEST4011 - a deletion derivative of the pEST4001. The restriction patterns of the plasmids mentioned above are considerably different from those of the other 2,4-D plasmids pJP4 and pRC10 reported previously.     

Structure of haloacetate-catabolic IncP-1beta plasmid pUO1 and genetic mobility of its residing haloacetate-catabolic transposon

The self-transmissible plasmid pUO1 from Delftia acidovorans strain B carries two haloacetate-catabolic transposons, TnHad1 and TnHad2, and the mer genes for resistance to mercury. The complete 67,066-bp sequence of pUO1 revealed that the mer genes were also carried by two Tn402/Tn5053-like transposons, Tn4671 and Tn4672, and that the pUO1 backbone regions shared 99% identity to those of the archetype IncP-1beta plasmid R751. Comparison of pUO1 with three other IncP-1beta plasmids illustrated the importance of transposon insertion in the diversity and evolution of this group of plasmids. Mutational analysis of the four outermost residues in the inverted repeats (IRs) of TnHad2, a Tn21-related transposon, revealed a crucial role of the second residue of its IRs in transposition.     

Bioaugmentation of activated sludge by an indigenous 3-chloroaniline-degrading Comamonas testosteroni strain, I2gfp

A strain identified as Comamonas testosteroni I2 was isolated from activated sludge and found to be able to mineralize 3-chloroaniline (3-CA). During the mineralization, a yellow intermediate accumulated temporarily, due to the distal meta-cleavage of chlorocatechol. This strain was tested for its ability to clean wastewater containing 3-CA upon inoculation into activated sludge. To monitor its survival, the strain was chromosomally marked with the gfp gene and designated I2gfp. After inoculation into a lab-scale semicontinuous activated-sludge (SCAS) system, the inoculated strain maintained itself in the sludge for at least 45 days and was present in the sludge flocs. After an initial adaptation period of 6 days, complete degradation of 3-CA was obtained during 2 weeks, while no degradation at all occurred in the noninoculated control reactor. Upon further operation of the SCAS system, only 50% 3-CA removal was observed. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes revealed a dynamic change in the microbial community structure of the activated sludge. The DGGE patterns of the noninoculated and the inoculated reactors evolved after 7 days to different clusters, which suggests an effect of strain inoculation on the microbial community structure. The results indicate that bioaugmentation, even with a strain originating from that ecosystem and able to effectively grow on a selective substrate, is not permanent and will probably require regular resupplementation.     

Conjugal transfer and characterization of bacteriocin plasmids in group N (lactic acid) streptococci.

Thirteen bacteriocin-producing strains of group N (lactic acid) streptococci were screened for their potential to transfer this property by conjugation to Streptococcus lactis subsp. diacetylactis Bu2-60. Bacteriocin production in three strains was plasmid encoded as shown by conjugal transfer and by analysis of cured, bacteriocin-negative derivatives of the donor strains and the transconjugants. With Streptococcus cremoris strains 9B4 and 4G6 and S. lactis subsp. diacetylactis 6F7 as donors, bacteriocin-producing transconjugants were isolated with frequencies ranging from ca. 2 X 10(-2) to 2 X 10(-1) per recipient cell. Bacteriocin-producing transconjugants had acquired a 39.6-megadalton plasmid from the donor strains 9B4 and 4G6, and a 75-megadalton plasmid from the donor strain 6F7. As shown by restriction endonuclease analysis, the plasmids from strains 9B4 and 4G6 were almost identical. The plasmid from strain 6F7 yielded some additional fragments not present in the two other plasmids. In hybridization experiments any of the three plasmids strongly hybridized with each other and with some other bacteriocin but nontransmissible plasmids from other S. cremoris strains. Homology was also detected to a variety of cryptic plasmids in lactic acid streptococci.     

Recipient Range of IncP-7 Conjugative Plasmid pCAR2 from Pseudomonas putida HS01 is Broader than from Other Pseudomonas Strains

The carbazole-degradative plasmid pCAR2 was isolated from Pseudomonas putida and had a genetic structure similar to that of pCAR1, the IncP-7 archetype plasmid. Mating analyses of pCAR2 with various recipient strains showed that it could transfer from HS01 to Pseudomonas recipients: P. chlororaphis, P. fluorescens, P. putida, P. resinovorans and P. stutzeri. The range of recipients changed when different hosts were used as a donor of pCAR2. The range of the plasmid from strain HS01 was broader than that using P. resinovorans CA10dm4 or P. putida KT2440. When pCAR1 or pCAR2 was transferred from the same cell background, the range and frequency of conjugation were now similar. Quantitative RT-PCR analyses indicated that tra/trh genes on both plasmids were similarly transcribed in each donor strain suggesting that the conjugative machinery of both plasmids may function similarly, and that other host factors are affecting the recipient range and frequency of conjugation.