Department of Microbiology, University of Georgia, Athens, GA 30602-2605, USA1
Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, GA 30602, USA2
Author for correspondence: Ellen L. Neidle. Tel: +1 706 542 2852. Fax: +1 706 542 2674. e-mail: eneidle{at}arches.uga.edu
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: porin, BenM, CatM, LTTR (LysR-type transcriptional regulators)
Abbreviations: CCM, cis,cis-muconate
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Upstream of benP are genes involved in the degradation of alkyl salicylates (salA, -R, -E, -D) and the degradation of alkanoate esters of benzyl alcohols (areA, -B, -C, -R) (Jones et al., 1999 , 2000
; Jones & Williams, 2001
). The SalE and AreABC enzymes convert various aromatic compounds to salicylate (2-hydroxybenzoate) or benzoate. Salicylate is the substrate of SalA, a hydroxylase that mediates the production of catechol. Genes for the degradation of catechol and those that enable benzoate to be converted to catechol are immediately downstream of benK (Fig. 1
) (Collier et al., 1998
). Thus, benP and benK are sandwiched between genes that help funnel a wide array of aromatic compounds into catechol, the substrate of a ring-cleaving dioxygenase (Fig. 1
). This genetic arrangement may reflect the participation of BenP and BenK in aromatic compound transport.
|
The function and expression of benP have not previously been investigated. Porin-like genes similar to benP have been identified near several genetic regions involved in bacterial aromatic compound degradation (Cowles et al., 2000 ; Segura et al., 1999
). For example, phaK, essential for the assimilation of phenylacetic acid in a Pseudomonas putida strain, appears to encode a specific channel-forming protein (Olivera et al., 1998
). The presence of genes likely to encode porins in different catabolic regions suggests that protein channels can facilitate the entry of aromatic compounds into Gram-negative bacteria. In ADP1, the location of the benP gene immediately upstream of benK suggested that both might be co-expressed. It also seemed likely that CatM and/or BenM would control gene expression. To test these possibilities, RT-PCR and primer-extension methods were used to study the transcriptional regulation of benP and benK. In addition, the CatM and BenM proteins were purified to enable further investigation of their roles in regulating the ß-ketoadipate pathway for aromatic compound dissimilation.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
To generate an Acinetobacter strain with an inactivated chromosomal benP gene, plasmid pBAC370 was digested with NdeI to yield a linear fragment with the disrupted benP::S5450 allele. This fragment was purified from a 1% agarose gel using the Qiaquick purification kit (Qiagen). As described previously (Neidle et al., 1989
), the linearized DNA fragment was used to transform and to replace the corresponding chromosomal region of strain ADP1, generating ACN450. The correct chromosomal configuration of ACN450 was confirmed by Southern hybridization analysis as previously described (Gregg-Jolly & Ornston, 1990
).
Expression and purification of CatM and BenM.
E. coli strain BL21-Gold (DE3) (Novagen) was transformed with pBAC381 or pBAC382 and plated on solid medium with ampicillin. An isolated colony was used to inoculate 5 ml LB broth with ampicillin, and the culture was grown at 37 °C with agitation until reaching an OD600 between 0·4 and 0·8. This 5 ml culture was used to inoculate 1 litre of fresh medium and allowed to grow to an OD600 of 0·40·8, at which point the culture was put on ice. After 10 min on ice, the culture was transferred to a 16 °C incubator, and protein expression was induced with the addition of IPTG to a final concentration of 1 mM. After overnight incubation (1014 h), each 1 litre culture was divided into 100 ml batches that were harvested by centrifugation at 7000 g for 10 min at 4 °C. After removal of the supernatant fluid, cell pellets were immediately stored at -70 °C.
Cell pellets containing CatM or BenM were suspended on ice in 10 ml buffer A1 (50 mM Tris/HCl pH 6·0, 50 mM NaCl, 5%, v/v, glycerol, 0·5 mM EDTA and 0·5 mM DTT) containing PMSF at a final concentration of 100 µg ml-1. Cells were lysed by two passages through a 4 °C chilled French pressure cell at 15000 p.s.i. (103·5 MPa). The resulting lysate was centrifuged at 15000 g for 15 min at 4 °C. All column purification steps were done with an FPLC system from Amersham-Pharmacia. A 10 ml sample of the supernate was loaded onto a 5 ml HITRAP SP cation-exchange column that had been equilibrated with 25 ml buffer A1. Protein was eluted from the column at a flow rate of 1 ml min-1 over a linear gradient of buffer B1 (buffer A1 with 1 M NaCl) and immediately placed on ice. Fractions containing CatM (300350 mM NaCl) or BenM (250350 mM NaCl) were pooled following analysis by 12% SDS-PAGE (Sambrook et al., 1989 ).
To lower the NaCl concentration and allow subsequent binding of CatM or BenM to additional columns, pooled samples were diluted fivefold with buffer A2 (50 mM Tris/HCl pH 7·0, 50 mM NaCl, 5%, v/v, glycerol, 0·5 mM EDTA and 0·5 mM DTT). The mixture was concentrated to a 10 ml volume with an Ultrafree S-10 centrifuge concentrator (Millipore). Samples were loaded onto a 5 ml HITRAP heparin-agarose affinity column that had been equilibrated with 25 ml buffer A2. Protein was eluted over a linear gradient of buffer B2 (Buffer A2 with 1 M NaCl) at 1 ml min-1. Fractions containing CatM (500600 mM NaCl) or BenM (300400 mM NaCl) were identified by SDS-PAGE. Samples containing pure protein were pooled, diluted fivefold with buffer A2, and concentrated to 13 ml using an Ultrafree S-10 centrifuge concentrator. Samples were stored in 20 µl aliquots at -70 °C, at a final concentration of 0·51·0 mg ml-1.
Protein concentrations were determined by the method of Bradford (1976) with bovine serum albumin as the standard. The molecular mass of CatM was estimated by gel-filtration chromatography. A 5 mg sample of purified CatM (1·0 mg ml-1) was loaded onto a HiPrep Sephacryl S-200 gel-filtration column that had been equilibrated with buffer A2. Protein was eluted and fractions analysed by SDS-PAGE to determine the elution volume for CatM. Known proteins served as standards according to the directions of the Molecular mass-Gel Filtration-200 kit (Sigma).
RNA extraction, RT-PCR, and primer-extension analysis.
Total RNA was extracted from mid-exponential-phase (OD600 0·30·5) Acinetobacter cultures by a hot phenol extraction method as described by Williams & Rogers (1987) . RNA samples were treated with DNase I (Promega). The Dnase I was later removed from the RNA with RNeasy columns (Qiagen) in accordance with the manufacturers specifications. Total RNA concentrations were estimated spectrophotometrically and rRNA bands were visualized on a 1·0% agarose gel (Sambrook et al., 1989
).
The Omniscript reverse transcriptase kit (Qiagen) was used for both RT-PCR and primer-extension methods. Taq polymerase (Fisherbrand) and RNasin ribonuclease inhibitor (Promega) were used for RT-PCR reactions. Products were analysed on 1·0% agarose gels and sometimes purified with the QIA-quick gel extraction kit (Qiagen). For primer-extension reactions, the BENP-PE primer (Table 2) was end-labelled with T4 polynucleotide kinase (Promega) and [
-32P]ATP (ICN Biomedicals) according to the manufacturers instructions. G-25 spin columns (Amersham-Pharmacia) were used to remove free [
-32P]ATP from the labelling reactions. One microlitre of the end-labelled primer was combined with 2 µg total RNA in a final volume of 6 µl and incubated at 65 °C for 5 min to allow primer annealing. A DNA sequencing ladder was generated with the fmol DNA Cycle Sequencing kit (Promega),
-32P end-labelled BENP-PE primer, and plasmid pBAC84 containing the wild-type benP region as template. Primer-extension products and sequencing reactions were analysed by 6% denaturing PAGE and visualized by autoradiography (Sambrook et al., 1989
).
Gel-retardation assays.
A double-stranded DNA fragment with the benP operatorpromoter region (PbP) was radiolabelled as previously described (Parsek et al., 1994 ). The BENPO/P-FOR and BENPO/P-REV primers, after being 5' end-labelled with [
-32P]ATP and T4 polynucleotide kinase (Promega), were used to PCR-amplify a 317 bp DNA fragment from pBAC84. This labelled fragment was purified following separation by 5% PAGE. The conditions for DNA-binding reactions were previously described (Romero-Arroyo et al., 1995
). Binding reactions were initiated by mixing 51000 ng CatM or BenM with 1000 c.p.m. of the 32P-labelled benP fragment (approx. 10 nM) in the presence or absence of 50 mM CCM. The DNAprotein complexes were analysed as described by Parsek et al. (1994)
.
Unlabelled DNA fragments were used in competition studies with the labelled fragment to verify the specificity of CatM and BenM binding. For use as a specific competitor, a DNA fragment with PbP was generated by PCR as described above except that the BENPO/P-FOR and BENPO/P-REV primers were not labelled with 32P. To generate a fragment for non-specific DNA competition studies, the M13 forward and M13 reverse universal primers (Promega) were used in PCR to amplify a 656 bp fragment from pBAC539 (Table 1), which contains the pobRA operatorpromoter region. This region of the ADP1 chromosome regulates the catabolism of p-hydroxybenzoate (DiMarco et al., 1993
) and does not contain a recognizable binding sequence for CatM or BenM. Competition studies were done by adding an increasing range of competitor DNA, from 1- to 50-fold molar excess, to the reaction as previously described (Tobiason et al., 1999
).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
RT-PCR analysis was carried out with the BENP-FOR primer in benP and the BENK-REV primer in benK (Fig. 2, Table 2
). With RNA from ADP1 grown on benzoate or CCM, but not succinate, a 1015 bp product of the size expected for these primers was obtained (Fig. 2b
). Control reactions lacking reverse transcriptase did not yield a product, indicating no significant DNA contamination in the RNA samples. RT-PCR products were isolated and digested with XbaI to confirm the presence of a known recognition sequence in benK. Although complete digestion was not always achieved, the sizes of the cleavage products were consistent with the correct fragments having been amplified, as shown for CCM-grown cells in Fig. 2(b)
, lane 7. Additional RT-PCR experiments with the same BENK-REV primer and a primer closer to the 5' end of benP (BENP2-FOR, Table 2
) yielded a product of the expected size with RNA from cells grown on CCM (data not shown). Thus, there were benK transcripts that initiate more than 700 nt upstream of its translational start codon. These results indicate that benP and benK are co-transcribed and that expression of the benPK operon occurs during growth on CCM or benzoate, but not succinate.
Transcript initiation and inducible expression of benPK
Expression of benP was further explored with primer-extension methods. The primer used for these experiments (BENP-PE, Table 2) annealed to a region downstream of the predicted benP AUG start codon. In extension reactions with total RNA from ADP1 grown on benzoate or CCM as the carbon source, two prominent products were generated. Their sizes would correspond to transcripts that initiate at sites 49 and 40 nt upstream of the start codon, labelled A and B in Fig. 3(a)
. No extension products were detected in reactions with RNA from succinate-grown cells, despite multiple repetitions with independently isolated RNA samples. Spectral analysis of total RNA and electrophoretic detection of rRNA revealed no obvious differences among the RNA samples from cells grown on different carbon sources. Consistent with the RT-PCR results, these studies indicated that benP expression is inducible.
|
Roles of CatM and BenM in regulating PbP
The identification of CCM as an inducer raised the possibility that CatM or BenM can activate transcription from PbP. To test this possibility, benP expression was studied in mutants (listed in Table 1) lacking CatM (strain ISA13), BenM (strain ISA36), or both of these transcriptional regulators (strain ACN9). Total RNA was isolated from the wild-type and from each of these mutants grown on rich medium supplemented with CCM. Primer-extension reactions with the benP primer (BENP-PE, Table 2
) indicated that the loss of both transcriptional regulators significantly reduced transcription from PbP (Fig. 4
). In contrast, the absence of either CatM or BenM alone had little effect on transcription relative to that in the wild-type strain under the same growth conditions (Fig. 4
). These results suggested that both BenM and CatM are individually able to regulate PbP in response to CCM. The overlap of their regulatory capabilities may account for the requirement that both BenM and CatM be absent in order for the loss of regulated gene expression to observed. A similar overlap in regulation by BenM and CatM occurs for the expression of catA from its promoter region PcA (Romero-Arroyo et al., 1995
).
|
|
|
The function of benP
The regulation of PbP by BenM and CatM should coordinate the transcription of the benPK operon with the expression of additional ben and cat genes involved in benzoate degradation (Fig. 1). Therefore, the ability to degrade benzoate and related compounds was tested in ACN450, a strain in which the chromosomal copy of benP was disrupted by insertional inactivation (Table 1
, Methods). ACN450 was capable of growing on solid medium using benzoate, benzaldehyde, benzyl alcohol, benzyl acetate, ethyl salicylate, salicylate or anthranilate as the sole carbon source (data not shown). With these substrates, possible differences in growth rates between ACN450 and the wild-type strain were not characterized. The mutation in ACN450 should also prevent expression of benK, a gene known to affect the rate of growth on some of these compounds (Collier et al., 1997
). These results indicate that neither BenP nor BenK is essential for the cellular entry of this set of aromatic compounds under the laboratory conditions tested. Different experimental conditions, different substrates and/or different genetic backgrounds may be needed to reveal the phenotypic effects caused by the loss of BenK and BenP.
Initial analysis of BenP detected relatively low, but significant, sequence identity in pairwise alignments with known porins. However, computer-based programs failed to optimize the alignment of BenP and other proteins throughout their entire lengths. As shown in Fig. 7, generated by a combination of computer-based methods and hand-made alignments, there are segments of BenP with strong sequence similarity to known or putative porins that are interspersed with regions, of variable size, containing little or no identity to the other sequences. According to the transporter classification (TC) system of Saier, BenP and several other putative porins involved in aromatic compound transport form a family that includes the OprD porin of Pseudomonas aeruginosa (TC# 1.B.25; http://tcdb.ucsd.edu/tcdb/search2.php) (Saier, 2000
). The substrates of OprD include cationic amino acids, peptides and an analogous compound, the antibiotic imipenem (Trias & Nikaido, 1990
).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This sequence alignment places the previously identified transcriptional start sites of the benA, catA and catB genes in an identical position (+1) (Fig. 1c) (Collier, 2000
; Romero-Arroyo et al., 1995
). This position of benP would correspond to a primer-extension product of the size denoted as A in Figs 3
and 4
. Thus, the placement of putative regulatory sequences and the observation that A is the largest observed primer-extension product suggest that benP transcription initiates at a position corresponding to that of the other CatM- and BenM-regulated genes. The multiple products in the benP primer-extension reactions may result from RNA secondary structure, RNA processing and/or degradation, multiple transcription initiation sites, or some combination of these factors. The significance of the experimental variation in relative intensities of the products is not evident. Nevertheless, the presence of the primer-extension products clearly indicated the conditions under which benP is expressed.
The regulon controlled by CatM and BenM
CCM caused transcription from PbP to increase in strains with CatM or BenM (Figs 3 and 4
). The absence of both regulators significantly reduced benP expression, although the resultant expression was higher than that of the wild-type strain grown in the absence of CCM (Fig. 4
). Similarly, in a mutant lacking both BenM and CatM, the expression of benA is approximately fourfold higher than in strains with the regulators when no inducer is present (Cosper et al., 2000
). This increased expression results from the ability of CatM or BenM, in the absence of inducers, to bind to a segment of PbA and repress basal benA expression by preventing access to RNA polymerase (Bundy, 2001
). BenM and CatM, which are able to bind to the PbP region in the absence of CCM (Fig. 6
), may similarly repress basal benP expression.
The gel-retardation studies indicate that CatM can form at least two different complexes with the PbP region. The presence of CCM may alter the interactions between the regulatory proteins and one or more binding site in this region. Multiple binding sites for BenM and CatM have been demonstrated in the PbA region (Bundy, 2001 ). Multiple binding sites in the target genes have also been identified in Pseudomonas putida for the CatR and ClcR regulators, which control catechol and chlorocatechol degradation, respectively (McFall et al., 1998
). Based on structural studies of transcriptional regulators, the putative CatM/BenM binding site in the PbP region, boxed in Fig. 1(c)
, should bind a protein dimer (Branden & Tooze, 1999
). However, BenM and CatM, like many LysR-type regulators, were tetrameric in solution (Schell, 1993
). Additional studies are needed to determine if the presence of CCM affects the oligomeric structure of CatM or BenM and to characterize the specific binding of these regulators to the PbP region.
While CatM and BenM both regulate transcription from PbP, PbA, PcA and PcB (depicted in Fig. 1), the specific regulation at each locus varies considerably. For example, benzoate interacts with BenM to activate transcription from PbA, and most likely from PcA, as well (Collier et al., 1998
; Romero-Arroyo et al., 1995
). In contrast, despite the ability of BenM to regulate transcription from PbP, benzoate did not serve as an inducer in the absence of its conversion to CCM (Figs 3
and 4
). Regulated expression from PbP appeared to be mediated equally well by CatM or BenM in response to CCM (Fig. 4
). Similarly, CCM enables CatM and BenM each to activate high-level transcription from PcA (Romero-Arroyo et al., 1995
). In contrast, at PbA or PcB, BenM or CatM, respectively, acts as the principal regulator of the expression of a multi-gene operon (Collier et al., 1998
; Romero-Arroyo et al., 1995
). Thus CatM and BenM control the expression of numerous genes with related functions. The demonstration in this report that the benPK operon is part of this complex regulon strongly suggests a role for BenP and BenK in the degradation of aromatic compounds.
Transporters in aromatic compound catabolic pathways
The proposed porin functions have yet to be demonstrated for BenP or for similar proteins likely to be involved in aromatic compound uptake. However, open reading frames encoding hypothetical bacterial porins are frequently located in the vicinity of genes known to participate in aromatic compound degradation. For example, two different BenP/PhaK-like hypothetical proteins of Pseudomonas sp. strain CA10 are encoded in genetic regions involved in the degradation of the heterocyclic aromatic compound carbazole via the catechol branch of the ß-ketoadipate pathway (Nojiri et al., 2001 ). Moreover, the putative porin-encoding genes are often located near genes encoding proteins resembling inner-membrane permeases such as BenK, PcaK and MucK that are involved in the uptake of compounds degraded via the ß-ketoadipate pathway (Nichols & Harwood, 1997
; Williams & Shaw, 1997
). Taken collectively, the data support a model in which a porin, such as BenP, and a permease, such as BenK, function together to facilitate the cellular entry of some substrates of bacterial aromatic compound catabolic pathways.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Branden, C. & Tooze, J. (1999). Introduction to Protein Structure, 2nd edn. New York: Garland Publishing.
Brzostowicz, P. C. (1997). Carbon-source dependent expression of the pobA gene needed for 4-hydroxybenzoate degradation in Acinetobacter sp. strain ADP1. MS thesis, University of Georgia.
Bundy, B. M. (2001). Transcriptional regulation of the benABCDE operon of Acinetobacter sp. strain ADP1: BenM-mediated synergistic induction in response to benzoate and cis,cis-muconate. PhD thesis, University of Georgia.
Coco, W. M., Parsek, M. R. & Chakrabarty, A. M. (1994). Purification of the LysR family regulator, ClcR, and its interaction with the Pseudomonas putida clcABD chlorocatechol operon promoter. J Bacteriol 176, 5530-5533.[Abstract]
Collier, L. S. (2000). Transcriptional regulation of benzoate degradation by BenM and CatM in Acinetobacter sp. strain ADP1. PhD thesis, University of Georgia.
Collier, L. S., Nichols, N. N. & Neidle, E. L. (1997). benK encodes a hydrophobic permease-like protein involved in benzoate degradation by Acinetobacter sp. strain ADP1. J Bacteriol 179, 5943-5946.[Abstract]
Collier, L. S., Gaines, G. L.III & Neidle, E. L. (1998). Regulation of benzoate degradation in Acinetobacter sp. strain ADP1 by BenM, a LysR-type transcriptional activator. J Bacteriol 180, 2493-2501.
Cosper, N. J., Collier, L. S., Clark, T. J., Scott, R. A. & Neidle, E. L. (2000). Mutations in catB, the gene encoding muconate cycloisomerase, activate transcription of the distal ben genes and contribute to a complex regulatory circuit in Acinetobacter sp. strain ADP1. J Bacteriol 182, 7044-7052.
Cowles, C. E., Nichols, N. N. & Harwood, C. S. (2000). BenR, a XylS homologue, regulates three different pathways of aromatic acid degradation in Pseudomonas putida. J Bacteriol 182, 6339-6346.
DiMarco, A. A., Averhoff, B. & Ornston, L. N. (1993). Identification of the transcriptional activator pobR and characterization of its role in the expression of pobA, the structural gene for p-hydroxybenzoate hydroxylase in Acinetobacter calcoaceticus. J Bacteriol 175, 4499-4506.[Abstract]
Gaines, G. L.III, Smith, L. & Neidle, E. L. (1996). Novel nuclear magnetic resonance spectroscopy methods demonstrate preferential carbon source utilization by Acinetobacter calcoaceticus. J Bacteriol 178, 6833-6841.[Abstract]
Gregg-Jolly, L. A. & Ornston, L. N. (1990). Recovery of DNA from the Acinetobacter calcoaceticus chromosome by gap repair. J Bacteriol 172, 6169-6172.[Medline]
Harwood, C. S. & Parales, R. E. (1996). The ß-ketoadipate pathway and the biology of self-identity. Annu Rev Microbiol 50, 553-590.[Medline]
Huang, H., Jenateur, D., Pattus, F. & Hancock, R. E. W. (1995). Membrane topology and site-specific mutagenesis of Pseudomonas aeruginosa porin OprD. Mol Microbiol 16, 931-941.[Medline]
Jones, R. M. & Williams, P. A. (2001). areCBA is an operon in Acinetobacter sp. strain ADP1 and is controlled by AreR, a 54-dependent regulator. J Bacteriol 183, 405-409.
Jones, R. M., Collier, L. S., Neidle, E. L. & Williams, P. A. (1999). areABC genes determine the catabolism of aryl esters in Acinetobacter sp. strain ADP1. J Bacteriol 181, 4568-4575.
Jones, R. M., Pagmantidis, V. & Williams, P. A. (2000). sal genes determining the catabolism of salicylate esters are part of a supraoperonic cluster of catabolic genes in Acinetobacter sp. strain ADP1. J Bacteriol 182, 2018-2025.
Juni, E. & Janik, A. (1969). Transformation of Acinetobacter calcoaceticus (Bacterium anitratum). J Bacteriol 98, 281-288.[Medline]
Keen, T., Tamaki, S., Kobayashi, D. & Trollinger, D. (1988). Improved broad-host-range plasmids for DNA cloning in Gram-negative bacteria. Gene 70, 191-197.[Medline]
Koebnik, R., Locher, K. P. & Van Gelder, P. (2000). Structure and function of bacterial outer-membrane proteins: barrels in a nutshell. Mol Microbiol 37, 239-253.[Medline]
McFall, S. M., Chugani, S. A. & Chakrabarty, A. M. (1998). Transcriptional activation of the catechol and chlorocatechol operons: variations on a theme. Gene 223, 257-267.[Medline]
Neidle, E. L., Hartnett, C. & Ornston, L. N. (1989). Characterization of Acinetobacter calcoaceticus catM, a repressor gene homologous in sequence to transcriptional activator genes. J Bacteriol 171, 5410-5421.[Medline]
Nichols, N. N. & Harwood, C. S. (1997). PcaK, a high-affinity permease for the aromatic compounds 4-hydroxybenzoate and protocatechuate from Pseudomonas putida. J Bacteriol 179, 5056-5061.[Abstract]
Nojiri, H., Sekiguchi, H., Maeda, K., Urata, M., Nakai, S., Yoshida, T., Habe, H. & Omori, T. (2001). Genetic characterization and evolutionary inplications of a car gene cluster in the carbazole degrader Pseudomonas sp. strain CA10. J Bacteriol 183, 3663-3679.
Ochs, M. M., Bains, M. & Hancock, R. E. W. (2000). Role of putative loops 2 and 3 in imipenem passage through the specific porin OprD of Pseudomonas aeruginosa. Antimicrob Agents Chemother 44, 1983-1985.
Olivera, E. R., Minambres, B., Garcia, B., Muniz, C., Moreno, M. A., Ferrandez, A., Diaz, E., Garcia, J. L. & Luengo, J. M. (1998). Molecular characterization of the phenylacetic acid catabolic pathway in Pseudomonas putida U: the phenylacetyl-CoA catabolon. Proc Natl Acad Sci USA 95, 6419-6424.
Parsek, M. R., Shinabarger, D. L., Rothmel, R. K. & Chakrabarty, A. M. (1992). Roles of CatR and cis,cis-muconate in activation of the catBC operon, which is involved in benzoate degradation in Pseudomonas putida. J Bacteriol 174, 7798-7806.[Abstract]
Parsek, M. R., Coco, W. M. & Chakrabarty, A. M. (1994). Gel-shift assay and Dnase I footprinting analysis of transcriptional regulation of biodegradation genes. Methods Mol Genet 3, 273-290.
Prentki, P. & Krisch, H. M. (1984). In vitro insertional mutagenesis with a selectable DNA fragment. Gene 29, 303-313.[Medline]
Romero-Arroyo, C. E., Schell, M. A., Gaines, G. L.III & Neidle, E. L. (1995). catM encodes a LysR-type transcriptional activator regulating catechol degradation in Acinetobacter calcoaceticus. J Bacteriol 177, 5891-5898.[Abstract]
Saier, M. H. (2000). Families of proteins forming transmembrane channels. J Membr Biol 175, 165-180.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Schell, M. A. (1993). Molecular biology of the LysR family of transcriptional regulators. Annu Rev Microbiol 47, 597-626.[Medline]
Segura, A., Bunz, P. V., DArgenio, D. A. & Ornston, L. N. (1999). Genetic analysis of a chromosomal region containing vanA and vanB, genes required for conversion of either ferulate or vanillate to protocatechuate in Acinetobacter. J Bacteriol 181, 3494-3504.
Shanley, M. S., Neidle, E. L., Parales, R. E. & Ornston, L. N. (1986). Cloning and expression of Acinetobacter calcoaceticus catBCDE genes in Pseudomonas putida and Escherichia coli. J Bacteriol 165, 557-563.[Medline]
Tobiason, D. M., Lenich, A. G. & Glasgow, A. C. (1999). Multiple DNA binding activities of the novel site-specific recombinase, Piv, from Moraxella lacunata. J Biol Chem 274, 9698-9706.
Trias, J. & Nikaido, H. (1990). Protein D2 channel of the Pseudomonas aeruginosa outer membrane has a binding site for basic amino acids and peptides. J Biol Chem 265, 15680-15684.
Williams, M. G. & Rogers, M. (1987). Expression of the arg genes of Escherichia coli during arginine limitation dependent upon stringent control of translation. J Bacteriol 169, 1644-1650.[Medline]
Williams, P. A. & Shaw, L. E. (1997). mucK, a gene in Acinetobacter calcoaceticus ADP1 (BD413) encodes the ability to grow on exogenous cis,cis-muconate as sole carbon source. J Bacteriol 179, 5935-5942.[Abstract]
Yanisch-Perron, C., Vieira, J. & Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103-119.[Medline]
Young, D. M., Parke, D., DArgenio, D. A., Smith, M. A. & Ornston, L. N. (2001). Evolution of a catabolic pathway. ASM News 67, 362-369.
Received 27 September 2001;
revised 21 November 2001;
accepted 23 November 2001.