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PubMed2004

Identification of a novel dioxygenase involved in metabolism of o-xylene, toluene, and ethylbenzene by Rhodococcus sp. strain DK17.

Rhodococcus sp. strain DK17 is able to grow on o-xylene, benzene, toluene, and ethylbenzene. DK17 harbors at least two megaplasmids, and the genes encoding the initial steps in alkylbenzene metabolism are present on the 330-kb pDK2. The genes encoding alkylbenzene degradation were cloned in a cosmid clone and sequenced completely to reveal 35 open reading frames (ORFs). Among the ORFs, we identified two nearly exact copies (one base difference) of genes encoding large and small subunits of an iron sulfur protein terminal oxygenase that are 6 kb apart from each other. Immediately downstream of one copy of the dioxygenase genes (akbA1a and akbA2a) is a gene encoding a dioxygenase ferredoxin component (akbA3), and downstream of the other copy (akbA1b and akbA2b) are genes putatively encoding a meta-cleavage pathway. RT-PCR experiments show that the two copies of the dioxygenase genes are operonic with the downstream putative catabolic genes and that both operons are induced by o-xylene. When expressed in Escherichia coli, AkbA1a-AkbA2a-AkbA3 transformed o-xylene into 2,3- and 3,4-dimethylphenol. These were apparently derived from an unstable o-xylene cis-3,4-dihydrodiol, which readily dehydrates. This indicates a single point of attack of the dioxygenase on the aromatic ring. In contrast, attack of AkbA1a-AkbA2a-AkbA3 on ethylbenzene resulted in the formation of two different cis-dihydrodiols resulting from an oxidation at the 2,3 and the 3,4 positions on the aromatic ring, respectively.

Benzene Derivatives↗

PubMed2005

Engineering Pseudomonas fluorescens for biodegradation of 2,4-dinitrotoluene.

Using the genes encoding the 2,4-dinitrotoluene degradation pathway enzymes, the nonpathogenic psychrotolerant rhizobacterium Pseudomonas fluorescens ATCC 17400 was genetically modified for degradation of this priority pollutant. First, a recombinant strain designated MP was constructed by conjugative transfer from Burkholderia sp. strain DNT of the pJS1 megaplasmid, which contains the dnt genes for 2,4-dinitrotoluene degradation. This strain was able to grow on 2,4-dinitrotoluene as the sole source of carbon, nitrogen, and energy at levels equivalent to those of Burkholderia sp. strain DNT. Nevertheless, loss of the 2,4-dinitrotoluene degradative phenotype was observed for strains carrying pJS1. The introduction of dnt genes into the P.fluorescens ATCC 17400 chromosome, using a suicide chromosomal integration Tn5-based delivery plasmid system, generated a degrading strain that was stable for a long time, which was designated RE. This strain was able to use 2,4-dinitrotoluene as a sole nitrogen source and to completely degrade this compound as a cosubstrate. Furthermore, P. fluorescens RE, but not Burkholderia sp. strain DNT, was capable of degrading 2,4-dinitrotoluene at temperatures as low as 10 degrees C. Finally, the presence of P. fluorescens RE in soils containing levels of 2,4-dinitrotoluene lethal to plants significantly decreased the toxic effects of this nitro compound on Arabidopsis thaliana growth. Using synthetic medium culture, P. fluorescens RE was found to be nontoxic for A.thaliana and Nicotiana tabacum, whereas under these conditions Burkholderia sp. strain DNT inhibited A.thaliana seed germination and was lethal to plants. These features reinforce the advantageous environmental robustness of P. fluorescens RE compared with Burkholderia sp. strain DNT.

Biodegradation, Environmental↗

PubMed2006

Genetic and genomic insights into the role of benzoate-catabolic pathway redundancy in Burkholderia xenovorans LB400.

Transcriptomic and proteomic analyses of Burkholderia xenovorans LB400, a potent polychlorinated biphenyl (PCB) degrader, have implicated growth substrate- and phase-dependent expression of three benzoate-catabolizing pathways: a catechol ortho cleavage (ben-cat) pathway and two benzoyl-coenzyme A pathways, encoded by gene clusters on the large chromosome (boxC) and the megaplasmid (boxM). To elucidate the significance of this apparent redundancy, we constructed mutants with deletions of the ben-cat pathway (the DeltabenABCD::kan mutant), the boxC pathway (the DeltaboxABC::kan mutant), and both pathways (the DeltabenABCDDelta boxABC::kan mutant). All three mutants oxidized benzoate in resting-cell assays. However, the DeltabenABCD::kan and DeltabenABCD DeltaboxABC::kan mutants grew at reduced rates on benzoate and displayed increased lag phases. By contrast, growth on succinate, on 4-hydroxybenzoate, and on biphenyl was unaffected. Microarray and proteomic analyses revealed that cells of the DeltabenABCD::kan mutant growing on benzoate expressed both box pathways. Overall, these results indicate that all three pathways catabolize benzoate. Deletion of benABCD abolished the ability of LB400 to grow using 3-chlorobenzoate. None of the benzoate pathways could degrade 2- or 4-chlorobenzoate, indicating that the pathway redundancy does not directly contribute to LB400's PCB-degrading capacities. Finally, an extensive sigmaE-regulated oxidative stress response not present in wild-type LB400 grown on benzoate was detected in these deletion mutants, supporting our earlier suggestion that the box pathways are preferentially active under reduced oxygen tension. Our data further substantiate the expansive network of tightly interconnected and complexly regulated aromatic degradation pathways in LB400.

Bacterial Proteins↗

PubMed2002

Characterization of a novel type IV pilus locus encoded on the large plasmid of locus of enterocyte effacement-negative Shiga-toxigenic Escherichia coli strains that are virulent for humans.

The majority of Shiga-toxigenic Escherichia coli (STEC) strains isolated from humans with gastrointestinal disease carry large (approximately 90-kb) plasmids. We have been analyzing the megaplasmid (designated pO113) from an O113:H21 STEC strain (98NK2). This strain lacks the locus for enterocyte effacement (LEE) and yet was responsible for an outbreak of hemolytic uremic syndrome. In the present study, we demonstrate that pO113 carries a novel type IV pilus biosynthesis locus (pil) related to those of the IncI plasmids R721, R64, and ColIb9. The pO113 pil locus consists of 11 closely linked genes (pilL through pilV) with an additional separately transcribed upstream gene (pilI). It directs the expression of long thin pili on the 98NK2 surface and the hemagglutination of guinea pig erythrocytes. We also demonstrate that pO113 can be transferred by conjugation. However, the type IV pilus encoded by pO113 does not appear to be involved in the adherence of 98NK2 to HEp-2 or Hct-8 cells in vitro. Homologues of the pO113 pil locus were present in several other LEE-negative STEC strains but not in LEE-positive STEC strains belonging to serogroup O26, O111, or O157.

Animals↗

PubMed2003

Enhanced CXC chemokine responses of human colonic epithelial cells to locus of enterocyte effacement-negative shiga-toxigenic Escherichia coli.

There is increasing evidence that by facilitating translocation of Shiga toxin (Stx) across the intestinal epithelium and by transporting bound toxin to remote sites such as the renal endothelium, polymorphonuclear leukocytes (PMNs) play a key role in the pathogenesis of Shiga-toxigenic Escherichia coli (STEC) disease. Plasma levels of PMN-attracting CXC chemokines such as interleukin-8 (IL-8) also appear to correlate in humans with the severity of disease. Thus, the capacity of STEC strains to elicit CXC chemokine responses in intestinal epithelial cells may be a crucial step in pathogenesis. Accordingly, we attempted to determine which STEC factors are responsible for CXC chemokine induction in human colonic epithelial cells. Infection of Hct-8 cells with locus for enterocyte effacement (LEE)-negative STEC strains isolated from patients with severe STEC disease resulted in up-regulation of IL-8, macrophage inflammatory protein 2alpha (MIP-2alpha), MIP-2beta, and ENA-78 mRNA significantly higher and earlier than that elicited by several LEE-positive STEC strains, including the O157:H7 strain EDL933. Similarly, levels of IL-8 protein in LEE-negative STEC-infected Hct-8 culture supernatants were significantly higher than in LEE-positive STEC-infected culture supernatants. The difference in responses could not be attributed to the expression or nonexpression of LEE genes, the presence or absence of an STEC megaplasmid, or differences in O serogroups or in the type or amount of Stx produced. Interestingly, however, several of the LEE-negative STEC strains eliciting the strongest chemokine responses belonged to flagellar serotype H21. Incubation of Hct-8 cells with isolated H21 flagellin elicited IL-8 and MIP-2alpha responses similar to those seen in the presence of the most potent LEE-negative STEC strains. Deletion of the fliC gene, but not the stx(2) gene, largely abolished the capacity of O113:H21 LEE-negative STEC strain 98NK2 to elicit IL-8 and MIP-2alpha responses in Hct-8 cells. Taken together, these data suggest that although Stx is capable of inducing CXC chemokine responses, the elevated responses seen in cells infected with certain STEC strains are largely attributable to the production of flagellin.

Base Sequence↗

PubMed2025

Quorum sensing and DNA methylation play active roles in clinical Burkholderia phase variation.

Phenotypic diversity in bacteria often results from adaptation to changing environmental conditions, exemplified by variable colony morphotypes. In Burkholderia pseudomallei, discrete genomic alterations and modulation of gene expression facilitate adaptation. Adapted variants of species within the Burkholderia cepacia complex (Bcc) often lose the pC3 virulence megaplasmid, impacting their colony morphology and their production of virulence factors. In this study, we characterize variants arising in Burkholderia ambifaria clinical isolates using proteomics and phenotypic tests and show that some of them have retained the pC3, indicating a distinct phase variation mechanism at play in this Bcc species. Interestingly, variants of B. ambifaria strains CEP0996 (pC3-null) and HSJ1 (pC3-positive) still share similarities in phenotypes controlled by the Cep quorum-sensing (QS) system. We further investigated the role of QS in B. ambifaria HSJ1 phase variation and confirmed that the Cep QS system is important for the emergence of variants. Given that DNA methylation is a key epigenetic factor regulating virulence factors in Burkholderia cenocepacia, we hypothesized that adenosine DNA methylation also governs phase variation in B. ambifaria HSJ1. By deleting the genes encoding putative adenosine DNA methyltransferases, we discovered that an orphan type II DNA methyltransferase inhibits the emergence of phase variants. This study is the first to demonstrate that quorum sensing and adenosine DNA methylation are two antagonistic systems independently controlling phase variation in B. ambifaria.IMPORTANCESome Burkholderia species are pathogenic to plants, animals, or humans. In immunocompromised individuals, and people suffering from cystic fibrosis, infection from the Burkholderia cepacia complex (Bcc) can lead to "cepacia syndrome." In northern Australia and southeast Asia, melioidosis caused by Burkholderia pseudomallei is prevalent among native population, particularly among people with diabetes, chronic lung or kidney disease or alcoholism. Burkholderia's phenotypic plasticity, including colony morphotype variation (CMV), enables rapid adaptation to diverse environments, enhancing its survival and pathogenicity. This study reveals phase variation as a new CMV mechanism within the Bcc group and is the first to report that quorum sensing and DNA methylation are involved in phase variation. Understanding the underlying mechanisms of CMV could lead to the development of targeted therapies against these highly antibiotic-tolerant bacteria.

Quorum Sensing↗

PubMed1984

Generalized transduction in Rhizobium meliloti.

Generalized transduction of Rhizobium meliloti 1021 was carried out by bacteriophage N3. Genetic markers on the chromosome and the pSym megaplasmid were transduced, along with markers on several IncP plasmids. Cotransduction between transposon Tn5 insertions and integrated recombinant plasmid markers permitted correlation of cotransductional frequencies and known physical distances. Bacteriophage N3 was capable of infecting several commonly used strains of R. meliloti.

Bacteriophages↗

PubMed1985

Alcaligenes eutrophus CH34 is a facultative chemolithotroph with plasmid-bound resistance to heavy metals.

Alcaligenes eutrophus strain CH34, which was isolated as a bacterium resistant to cobalt, zinc, and cadmium ions, shares with A. eutrophus strain H16 the ability to grow lithoautotrophically on molecular hydrogen, to form a cytoplasmic NAD-reducing and a membrane-bound hydrogenase, and most metabolic attributes; however, it does not grow on fructose. Strain CH34 contains two plasmids, pMOL28 (163 kilobases) specifying nickel, mercury, and cobalt resistance and pMOL30 (238 kilobases) specifying zinc, cadmium, mercury, and cobalt resistance. The plasmids are self-transmissible in homologous matings, but at low frequencies. The transfer frequency was strongly increased with IncP1 plasmids RP4 and pUZ8 as helper plasmids. The phenotypes of the wild type, cured strains, and transconjugants are characterized by the following MICs (Micromolar) in strains with the indicated phenotypes: Nic+, 2.5; Nic-, 0.6; Cob+A, 5.0; Cob+B, 20.0; Cob-, less than 0.07; Zin+, 12.0; Zin-, 0.6; Cad+, 2.5; and Cad-, 0.6. Plasmid-free cells of strain CH34 are still able to grow lithoautotrophically and to form both hydrogenases, indicating that the hydrogenase genes are located on the chromosome, in contrast to the Hox structural genes of strain H16, which are located on the megaplasmid pHG1 (450 kilobases).

Alcaligenes↗

PubMed1985

Physical and genetic map of a Rhizobium meliloti nodulation gene region and nucleotide sequence of nodC.

Infection of alfalfa by the soil bacterium Rhizobium meliloti proceeds by deformation of root hairs and bacterial invasion of host tissue by way of an infection thread. We studied an 8.7-kilobase (kb) segment of the R. meliloti megaplasmid, which contains genes required for infection. Site-directed Tn5 mutagenesis was used to examine this fragment for nodulation genes. A total of 81 R. meliloti strains with mapped Tn5 insertions in the 8.7-kb fragment were evaluated for nodulation phenotype on alfalfa plants; 39 of the insertions defined a 3.5-kb segment containing nodulation functions. Of these 39 mutants, 37 were completely nodulation deficient (Nod-), and 2 at the extreme nif-distal end were leaky Nod-. Complementation analysis was performed by inoculating plants with strains carrying a genomic Tn5 at one location and a plasmid-borne Tn5 at another location in the 3.5-kb nodulation segment. Mutations near the right border of the fragment behaved as two distinct complementation groups. The segment in which these mutations are located was analyzed by DNA sequencing. Several open reading frames were found in this region, but the one most likely to function is 1,206 bases long, reading from left to right (nif distal to proximal) and spanning both mutation groups. The genetic behavior of this segment may be due either to the gene product having two functional domains or to a recombinational hot spot between the apparent complementation groups.

Amino Acid Sequence↗

PubMed1985

Denitrification by Alcaligenes eutrophus is plasmid dependent.

Curing of the hydrogenase-specifying megaplasmid pHG indigenous to strains of the facultative lithoautotrophic bacterium Alcaligenes eutrophus was correlated with a loss of denitrifying ability (Nitd). The retransfer of plasmid pHG1 reconstituted the Nitd phenotype. Plasmid-free mutants were still capable of converting some nitrate to nitrite, but they did not metabolize nitrite under anaerobic conditions.

Alcaligenes↗

PubMed1985

Chromosomal and plasmid locations for phosphoribulokinase genes in Alcaligenes eutrophus.

Genes coding for phosphoribulokinase (PRK), a key enzyme of the Calvin cycle, were localized in the genome of the chemoautotroph Alcaligenes eutrophus. The NH2-terminal sequence of the PRK subunit was determined. With a synthetic oligodeoxynucleotide probe complementary to a portion of this sequence, hybridization analysis revealed PRK genes to be located on both the chromosome and the megaplasmid pHG1 of A. eutrophus H16.

Alcaligenes↗

PubMed1985

Identification of a Rhizobium meliloti pSym2011 region controlling the host specificity of root hair curling and nodulation.

In Rhizobium meliloti 2011 nodulation genes (nod) required to nodulate specifically alfalfa are located on a pSym megaplasmid. Nod- derivatives carrying large pSym deletions were isolated. By complementation of these strains with in vivo- and in vitro-constructed episomes containing pSym of sequences and introduction of these episomes into Agrobacterium tumefaciens, we show (i) that from a region of pSym of about 360 kilobases, genes required for specific alfalfa nodulation are clustered in a DNA fragment of less than 30 kilobases and (ii) that a nod region located between nifHDK and the common nod genes is absolutely required for alfalfa nodulation and controls the specificity of root hair curling and nodule organogenesis initiation.

Chromosome Deletion↗

PubMed1986

Two gene clusters of Rhizobium meliloti code for early essential nodulation functions and a third influences nodulation efficiency.

A pLAFR1 cosmid clone (pPP346) carrying the nodulation region of the symbiotic plasmid pRme41b was isolated from a gene library of Rhizobium meliloti 41 by direct complementation of a Nod- deletion mutant of R. meliloti. Agrobacterium tumefaciens and Rhizobium species containing pPP346 were able to form ineffective nodules on alfalfa. The 24-kilobase insert in pPP346 carries both the common nodulation genes and genes involved in host specificity of nodulation. It was shown that these two regions are essential and sufficient to determine the early events in nodulation. A new DNA region influencing the kinetics and efficiency of nodulation was also localized on the symbiotic megaplasmid at the right side of the nif genes.

DNA, Bacterial↗

PubMed1986

Molecular cloning of structural and regulatory hydrogenase (hox) genes of Alcaligenes eutrophus H16.

A gene bank of the 450-kilobase (kb) megaplasmid pHG1 from the hydrogen-oxidizing bacterium Alcaligenes eutrophus H16 was constructed in the broad-host-range mobilizable vector pSUP202 and maintained in Escherichia coli. hox DNA was identified by screening the E. coli gene bank for restoration of hydrogenase activity in A. eutrophus Hox mutants. Hybrid plasmids that contained an 11.6-kb EcoRI fragment restored soluble NAD-dependent hydrogenase activity when transferred by conjugation into one class of Hos- mutants. An insertion mutant impaired in particulate hydrogenase was partially restored in Hop activity by an 11-kb EcoRI fragment. A contiguous sequence of two EcoRI fragments of 8.6 and 2.0 kb generated Hox+ recombinants from mutants that were devoid of both hydrogenase proteins. hox DNA was subcloned into the vector pVK101. The resulting recombinant plasmids were used in complementation studies. The results indicate that we have cloned parts of the structural genes coding for Hos and Hop activity and a complete regulatory hox DNA sequence which encodes the thermosensitive, energy-dependent derepression signal of hydrogenase synthesis in A. eutrophus H16.

Alcaligenes↗

PubMed1986

Assignment of symbiotic developmental phenotypes to common and specific nodulation (nod) genetic loci of Rhizobium meliloti.

Rhizobium meliloti nodulation (nod) genes required for specific infection and nodulation of alfalfa have been cloned. Transposon Tn5 mutagenesis defined three nod regions spanning 16 kilobases of the pSym megaplasmid. Genetic and cytological studies of 62 nodulation-defective mutants allowed the assignment of symbiotic developmental phenotypes to common and specific nod loci. Root hair curling was determined by both common (region I) and specific (region III) nod transcription units; locus IIIb (nodH gene) positively controlled curling on the homologous host alfalfa, whereas loci IIIa (nodFE) and IIIb (nodH) negatively controlled curling on heterologous hosts. Region I (nodABC) was required for bacterial penetration and infection thread initiation in shepherd's crooks, and the nodFE transcription unit controlled infection thread development within the alfalfa root hair. In contrast, induction of nodule organogenesis, which can be triggered from a distance, seemed to be controlled by common nodABC genes and not to require specific nod genes nodFE and nodH. Region II affected the efficiency of hair curling and infection thread formation.

Cloning, Molecular↗

PubMed1987

Rhizobium meliloti insertion element ISRm2 and its use for identification of the fixX gene.

Two of the three plasmids of the wild-type Rhizobium meliloti 41 (pRme41a and pRme41c) carry a copy of ISRm2, a 2.7-kilobase-long transposable element. ISRm2 is terminated by 22-base-pair (bp) inverted repeat sequences, exhibiting some homology to the inverted repeats of elements generating 9-bp target sequence duplication. Transposition of ISRm2 results in a duplication of 8 bp in length, rather rare among transposable elements. DNA sequences homologous to an internal fragment of ISRm2 were found in several Rhizobium species. Transposition of ISRm2 into fixation and nodulation genes located on the symbiotic plasmid pRme41b was detected at a high frequency. Exact locations of two copies of ISRm2 which transposed into the nod-nif region on the megaplasmid were determined. In one case, integration into the protein-coding region of the hsnD gene that determines a host specificity function of nodulation occurred. In the other mutant, ISRm2 was localized upstream of nifA, where a short open reading frame coding for a new fix gene (fixX) was identified. The product of fixX is a ferredoxin carrying a characteristic cluster of cysteine residues. On the basis of the observation that the arrangement of the ISRm2 copies is identical in the free-living wild-type cells and in nitrogen-fixing nodules, we concluded that the involvement of ISRm2 transposition in the development of nitrogen-fixing symbiosis is unlikely.

Cloning, Molecular↗

PubMed1988

Genetic and physical analyses of group E exo- mutants of Rhizobium meliloti.

Mutants of Rhizobium meliloti which are deficient in exopolysaccharide synthesis have been classified into six different genetic groups (A through F) (J. A. Leigh, E. R. Signer, and G. C. Walker, Proc. Natl. Acad. Sci. USA 82:6231-6235, 1985). Using physical and genetic techniques, we have demonstrated that the group E Exo- mutants carry deletions in the exoA-exoB region of the megaplasmid pRmeSU47b. We have constructed strains carrying defined deletions which remove up to 200 kilobases of pRmeSU47b, including the exoA-exoB region. These derivatives have the same phenotypes as do the group E mutants.

Base Sequence↗

PubMed1988

Chemotaxis of Rhizobium meliloti to the plant flavone luteolin requires functional nodulation genes.

Luteolin is a phenolic compound from plants that acts as a potent and specific inducer of nodABC gene expression in Rhizobium meliloti. We have found that R. meliloti RCR2011 exhibits positive chemotaxis towards luteolin. A maximum chemotactic response was observed at 10(-8) M. Two closely related flavonoids, naringenin and apigenin, were not chemoattractants. The presence of naringenin but not apigenin abolished chemotaxis of R. meliloti towards luteolin. A large deletion in the nif-nod region of the symbiotic megaplasmid eliminated all chemotactic response to luteolin but did not affect general chemotaxis, as indicated by swarm size on semisoft agar plates and chemotaxis towards proline in capillary tubes. Transposon Tn5 mutations in nodD, nodA, or nodC selectively abolished the chemotactic response of R. meliloti to luteolin. Agrobacterium tumefaciens GMI9050, a derivative of the C58 wild type lacking a Ti plasmid, responded chemotactically to 10(-8) M luteolin. The introduction of a 290-kilobase nif-nod-containing sequence of DNA from R. meliloti into A. tumefaciens GMI9050 enabled the recipient to respond to luteolin at concentrations peaking at 10(-6) M as well as at concentrations peaking at 10(-8) M. The response of A. tumefaciens GMI9050 to luteolin was also abolished by the presence of naringenin.

Chamomile↗