Sequence and genome context analysis of a new molecular class D ß-lactamase gene from Legionella pneumophila

Matthew B. Avison1,* and Alan M. Simm2

Bristol Centre for Antimicrobial Research and Evaluation (BCARE), Departments of 1 Biochemistry and 2 Pathology & Microbiology, University of Bristol, School of Medical Sciences, University Walk, Bristol BS8 1TD, UK

Received 8 February 2002; returned 26 April 2002; revised 2 May 2002; accepted 5 June 2002


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Legionella pneumophila Philadelphia-1 (ATCC 33152) produces a serine active site ß-lactamase. The chromosomal gene that encodes this enzyme, loxA, has been cloned by PCR using information from the L. pneumophila Philadelphia-1 genome sequencing project. LoxA is a class 2d penicillinase, and its sequence puts it into the molecular class D ß-lactamase family, although phylogenetic analysis shows that LoxA forms a distinct branch in the OXA family along with the LoxA homologue, OXA-29, from Legionella gormanii ATCC 33297T. Upstream of loxA on the L. pneumophila Philadelphia-1 chromosome is a two-gene locus similar to that found linked to the ß-lactamase genes of Gram-positive bacteria. The unit consists of loxI, encoding a homologue of the Gram-positive ß-lactamase expression regulator, and pbpX, encoding a putative penicillin-binding transpeptidase. Despite the presence of ß-lactamase regulator homologues, we could find no evidence of LoxA induction upon challenge of L. pneumophila Philadelphia-1 with ß-lactams.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The genus Legionella represents a wide variety of environmental organisms that can, under certain circumstances, cause pneumonia, particularly in debilitated individuals. By far the most common species of Legionella responsible for such infections is Legionella pneumophila. The fatality rate for Legionella pneumonia can be as high as 50% in immunocompromised patients, but if diagnosed early, antibiotic therapy commonly results in a successful outcome.1 In general, L. pneumophila clinical isolates, with L. pneumophila Philadelphia-1 being one of the most widely studied, do not display high levels of innate resistance to antibiotics, but it has long been known that they are almost ubiquitously resistant to penicillins in vivo, despite having low MICs of ß-lactams in vitro.26 Penicillin resistance in L. pneumophila Philadelphia-1 is believed to be due to the production of a single ß-lactamase with a pI > 8.0 and a molecular weight of ~30 kDa, which hydrolyses penicillins and nitrocefin, but not cephalosporins, and which is inhibited by serine ß-lactamase inhibitors.3,57 The sequence of the gene encoding this enzyme has not been reported previously.

The related bacterium Legionella gormanii shows much broader resistance to ß-lactams than L. pneumophila,7 and is known to produce a metallo-ß-lactamase, FEZ-1, which accounts for resistance to cephalosporins and, to a lesser extent, carbapenems.810 FEZ-1 does not hydrolyse penicillins,10 yet L. gormanii isolates show similar levels of resistance to these agents to L. pneumophila isolates.6,7 In a recent report, the gene blaOXA-29 was cloned from L. gormanii ATCC 33297T. It appears that this gene encodes the second ß-lactamase of L. gormanii, accounting primarily for resistance to penicillins.11 The aims of the project described in this report were to analyse the product(s), genetic location(s) and level(s) of expression of the ß-lactamase gene(s) from L. pneumophila Philadelphia-1 using information about L. gormanii ATCC 33297T ß-lactamases as a starting point. L. pneumophila Philadelphia-1 was chosen as the study organism because its genome is currently being sequenced at the Columbia Genome Center, NY, USA.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains

L. pneumophila serotype 1 (Philadelphia) (ATCC 33152)12 was obtained from the ATCC. It was plated onto charcoal yeast extract (CYE) agar and grown in 1% w/v yeast extract broth. In all cases, the medium was enriched with ‘Legionella BCYE’ growth supplement according to the manufacturer’s instructions (Oxoid, Basingstoke, UK). Escherichia coli DH5{alpha}13 was used as recipient for cloning and was grown in nutrient broth or on nutrient agar (Oxoid). In both cases, bacteria were grown at 37°C in air.

Materials

ß-Lactams used were nitrocefin (Beckton-Dickinson, Cockeysville, MD, USA); ceftazidime, clavulanic acid and BRL 42715 (GlaxoSmithKline, Worthing, UK); ampicillin, carbenicillin, cefaloridine, cefalothin and oxacillin (Sigma Chemical Co., St Louis, MO, USA); piperacillin (Lederle, Carolina, Puerto Rico); imipenem (Merck, Sharpe & Dohme, West Point, PA, USA); and meropenem (Zeneca Pharmaceuticals, Macclesfield, UK). PCR primers were purchased from Sigma-Genosys Ltd (Pampisford, UK). General reagents for DNA manipulation were obtained from Gibco-BRL (Life Technology Ltd, Paisley, UK). All other general reagents were from Sigma Chemical Co. or BDH (both Poole, UK).

PCR and cloning of PCR products

Genomic PCR was performed as described previously.14 The primers used to amplify L. pneumophila loxA were (forward) 5'-GGATGCTCTGAGCAGTGC-3' and (reverse) 5'-AGTGACGAGCTACTTAAC-3'. The primers were designed to allow sequencing of the entire coding sequence of the loxA gene, without the risk of the primer sequence affecting the observed loxA sequence. PCR amplicons from three separate PCRs were purified using a QIAquick PCR purification kit (Qiagen Ltd, Crawley, UK) according to the manufacturer’s instructions, and sequenced on both strands using the PCR primers to initiate sequencing. The loxA PCR amplicon was TA-cloned into the pCR4.1 cloning vector according to the manufacturer’s instructions (Invitrogen, Leek, The Netherlands) and the cloned insert was removed by EcoRI digestion and subcloned15 into EcoRI-linearized cloning vector pZero-2 (Invitrogen). The loxA PCR amplicon includes a putative loxA ribosome-binding sequence,16 and this approach allows translation of LoxA in E. coli from an mRNA in which the loxA transcript is fused to that of lacZ. Transcription of this fusion mRNA is driven from the ptac promoter of pZero-2 following IPTG induction. The appropriately oriented pZero-2 recombinant was selected and denoted pUB6059, and was used to transform E. coli DH5{alpha} to kanamycin resistance (30 mg/L).

In silico sequence analysis

Computer-assisted sequence manipulation and basic alignments were with the Lasergene software package (DNA Star, Madison, WI, USA). Genome sequence database interrogation and phylogenetic analysis of published molecular class D ß-lactamase protein sequences were performed as described previously.17

Induction of ß-lactamase expression, and isolation and assay of ß-lactamases

When using L. pneumophila Philadelphia-1, induction of ß-lactamase expression by ß-lactam challenge was attempted as described previously.18 More specifically, the Legionella liquid growth medium, defined above, was used, and numerous induction conditions were tested [cefoxitin, ampicillin or imipenem as inducer, each used at 0.1, 1.0, 10 or 100 mg/L (0.01–1000 x MIC,6 dependent upon the drug) for 2, 4 or 10 h]. When using E. coli DH5{alpha} carrying pUB6059 (loxA), expression of LoxA was induced by the addition of 0.1 mM (final) IPTG to a nutrient broth culture (OD600 0.6) for 2 h. In either case, cells were collected by centrifugation and crude cell extracts were prepared as described previously.18 Hydrolysis of ß-lactam antibiotics was examined by spectrophotometric analysis as described previously18 at the following wavelengths: 233, 235, 265, 265, 262, 299, 299, 482, 250 and 233 nm, and using the following extinction coefficients: –809, –830, –6980, –10781, –9000, –7000, –2500, +17400, +240 and –936 M–1·cm–1, respectively, for ampicillin, carbenicillin, cefaloridine, cefalothin, ceftazidime, imipenem, meropenem, nitrocefin, oxacillin and piperacillin. The protein concentration of each bacterial extract was determined using the Bio-Rad protein assay reagent (Bio-Rad, München, Germany) according to the manufacturer’s instructions. One unit of ß-lactamase activity was defined as that required to hydrolyse 1 µmol of substrate per minute at 25°C. Specific activity was defined as the number of units of ß-lactamase per mg of protein in the cell extract.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
PCR cloning of loxA, encoding a molecular class D penicillinase from L. pneumophila Philadelphia-1

Crude cell extracts were made from broth cultures of L. pneumophila Philadelphia-1, and ß-lactamase assays confirmed that these extracts contained an activity that could hydrolyse penicillins and nitrocefin, but not carbapenems or cephalosporins. The hydrolytic profile was indicative of a class 2d oxacillinase (Table 1).19 Whilst this activity was completely inhibited by 10 µM BRL 42715 and partially inhibited by 10 µM clavulanic acid (serine ß-lactamase inhibitors), it was insensitive to treatment with 50 mM EDTA (known to inhibit metallo-ß-lactamases).


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Table 1.  ß-Lactamase activities found in extracts of L. pneumophila isolate Philadelphia-1 and E. coli DH5{alpha} expressing L. pneumophila LoxA
 
A search for a homologue(s) of the L. gormanii blaOXA-29 serine ß-lactamase gene11 in the genome sequence of L. pneumophila Philadelphia-1 (http://genome3.cpmc.columbia.edu/~legion/int_blast.html) using an ungapped BLASTN search with the filter off revealed one significant hit. The DNA sequence of this locus was present on contig 267 (BC.3E47G7.26.24-R.091401) and was used to design PCR primers that would allow amplification of the gene from L. pneumophila Philadelphia-1 genomic DNA. Genomic PCR was successful and the resultant PCR amplicon was cloned into pZero-2 to produce pUB6059 as described in Materials and methods. Cellular extracts from DH5{alpha}:pUB6059 contained an almost identical profile of ß-lactamase activity to that seen when analysing L. pneumophila Philadelphia-1 crude extracts (Table 1). As such, it was thought highly likely that pUB6059 encoded the ß-lactamase activity present in extracts of L. pneumophila Philadelphia 1; therefore, the cloned ß-lactamase gene was designated loxA (for Legionella oxacillinase).

Sequence analysis of loxA from L. pneumophila Philadelphia-1

The L. pneumophila loxA gene extends for 801 nucleotides, encoding a peptide of 266 amino acids. A credible ribosome binding site is located nine nucleotides upstream from the initiation codon.16 No obvious promoter with significant homology to the {sigma}70 promoter of E. coli has been identified directly upstream of loxA (Figure 1).20 An alanine residue at position 20 and a glutamine at position 21 define a credible 20 amino acid secretion leader peptide21 in LoxA. The mature 246 amino acid protein has a predicted molecular weight of 28.6 kDa and a predicted pI of 9.4. LoxA has 76.5% identity to OXA-29 from L. gormanii ATCC 33297T.11 The LoxA protein sequence was aligned with all previously published examples of molecular class D ß-lactamases (Figure 2).22 This information was used to construct a phylogenetic tree for the molecular class D ß-lactamase family (Figure 3).



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Figure 1. The nucleotide and deduced amino acid sequence of L. pneumophila LoxA. The putative ribosome binding site is double underlined. The amino acids potentially involved in signal peptide cleavage are bold, and the cleavage site is marked with an asterisk.

 


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Figure 2. Alignment of published molecular class D ß-lactamase amino acid sequences. Multiple alignment of class D ß-lactamase proteins as described in Materials and methods. Regions of identity are shaded in black and regions of similarity are shaded grey. EMBL accession numbers for the published sequences are: OXA 1, P13661; OXA-2, P05191; OXA-3, Q51429; OXA-5, Q00982; OXA-7, P35695; OXA-9, P22070; OXA-10, P14489; OXA-11, Q06778; OXA-12, I39695, OXA-13, AAC46344; OXA-14, AAA93528; OXA-15, Q51574; OXA-18, O07293; OXA-19, AAD02245; OXA-20, O84955; OXA-21, P94124; OXA-23 (ARI-1), CAB69042; OXA-24, CAB92323; OXA-25,26,27, A201826,7,8; OXA-28, AAF72942; OXA-29, CAC35728; OXA-30, AAF72981; OXA-31, AAK52604; SLR0319, BAA10689; MLL0916, BAB48403; AmpS, CAA56560; AmpH, CAC01289; LCR-1, Q00983; PA5514, AAG08899; CJ0299, CAB72766; YbxI, P54427.

 


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Figure 3. Phylogenetic tree based on comparison of published class D ß-lactamase amino acid sequences. The alignment shown in Figure 2 was analysed as described in Materials and methods to produce a phylogenetic tree of the class D ß-lactamase amino acid sequences. Each number represents a node of sequence divergence and the length of the branch leading to the node represents the estimated number of substitutions per 100 amino acids along that branch. Where the software could not resolve differences between ß-lactamase amino acid sequences, the sequences were grouped and one terminal node represents each group. The ‘L’ value,17 representing the likelihood that this tree is the best description of the aligned sequences, is –10 039 ± 276. This is an unrooted tree.

 
Genome location and inducibility of loxA in L. pneumophila Philadelphia-1

Further analysis of contig 267 of the L. pneumophila Philadelphia-1 genome sequence revealed the genomic location of loxA (Figure 4). Interestingly, loxA is immediately downstream of pbpX, encoding a putative penicillin-binding protein having 27% identity to E. coli PBP-3. Further upstream is the gene loxI, encoding a homologue of the Gram-positive ß-lactamase transcriptional regulator BlaI (25% identical to BlaI from Bacillus licheniformis). This arrangement of a transcriptional regulator gene, a penicillin-binding protein-like gene and a ß-lactamase gene is associated with Gram-positive ß-lactamase genes that are induced during ß-lactam challenge of cells (Figure 4).23 Accordingly, it was thought possible that LoxA expression in L. pneumophila Philadelphia-1 might be inducible upon ß-lactam challenge through the actions of LoxI and PbpX. Using L. pneumophila Philadelphia-1, numerous induction conditions were tested (cefoxitin, ampicillin or imipenem as inducer, each used at 0.1, 1.0, 10 or 100 mg/L for 2, 4 or 10 h), but in no case could increased levels of ampicillin hydrolysing activity be detected in crude cell extracts.



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Figure 4. Organization of the loxA locus on the L. pneumophila Philadelphia-1 chromosome and comparison with the bla locus of Gram-positive bacilli.23 See text for details of the assignment of gene names. The predicted transcriptional orientation of each gene is marked with an arrow.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It has long been known that various members of the Legionellaceae express ß-lactamase enzymes.28 In this study, we report the cloning and basic characterization of a molecular class D, functional class 2d penicillinase from L. pneumophila Philadelphia-1, named LoxA, which has a predicted pI of 9.4, a molecular weight of 28.6 kDa, and appears to account for the entire ß-lactam hydrolytic profile found in extracts from the host organism (Table 1). These predictions are consistent with the characteristics of the only ß-lactamase previously detected in L. pneumophila Philadelphia-1. Whilst in parallel experiments we found no significant homologues of other classes of ß-lactamase in the L. pneumophila Philadelphia-1 genome sequence, we have not entirely excluded the possibility that other ß-lactamase genes are present, because ~10% of the genome remains to be sequenced. If such genes are present, however, they are not expressed at detectable levels (Table 1).7

The ß-lactam resistance profile of L. gormanii appears different to those of L. pneumophila isolates,6,7 and the ß-lactamase activity purified from L. gormanii strain ATCC 33297T has, in the past, been solely attributed to a metallo-ß-lactamase.8 However, whilst serine ß-lactamase inhibitors were shown to have no effect on cephalosporin hydrolysis (when EDTA completely abolished activity) their effect on the hydrolysis of penicillins was not reported.8 This is particularly relevant given that the gene encoding the metallo-ß-lactamase of L. gormanii ATCC 33297T, blaFEZ-1, has since been cloned,9 and detailed biochemical analysis of its product showed it to be a good cephalosporinase, but a poor penicillinase.10 Extracts of L. gormanii ATCC 33297T (and of other L. gormanii isolates) express almost equal amounts of penicillinase and cephalosporinase activity,7,8 so FEZ-1 cannot account for both these activities. Recently, however, a loxA homologue from L. gormanii ATCC 33297T, blaOXA-29, has been cloned and the OXA-29 enzyme characterized biochemically.11 It is predominantly a penicillinase, and so accounts for the penicillinase activity of L. gormanii ATCC 33297T extracts. LoxA and OXA-29 are ~75% identical.

The genetic context of loxA in L. pneumophila Philadelphia-1 reveals the presence of linked genes that are homologous, both in sequence and gene arrangement, to Gram-positive ß-lactamase regulatory genes. However, we could not detect the induction of LoxA expression in the presence of ß-lactams in L. pneumophila Philadelphia-1 using conditions known to induce ß-lactamase expression in bacteria with inducible ß-lactamases. It might be that the presence of those genes linked to loxA is by chance, and has no functional significance. Alternatively, this gene context may reveal a link between some aspects of L. pneumophila physiology, and the expression of LoxA. Interestingly, Plesiomonas shigelloides strains have been isolated that encode a chromosomal class 2d ß-lactamase, which is not induced in response to ß-lactam challenge,24 but might be induced in response to some other signal.25 Work is ongoing to confirm whether, in P. shigelloides, the ß-lactamase gene is in a similar genetic context to loxA in L. pneumophila Philadelphia-1.


    Acknowledgements
 
We are grateful to Pippa Harris (funded by a BSAC vacation scholarship) for technical help, and to Jennie Douthwaite, Department of Biochemistry, University of Bristol, for DNA sequencing. The L. pneumophila Philadelphia-1 genome sequence was obtained from the Columbia Genome Center, Columbia University, NY, USA. Sequencing of this genome was funded by the NIH. ß-Lactamase research at the Bristol Centre for Antimicrobial Research and Evaluation (BCARE) is supported by the BSAC and the Biotechnology and Biological Sciences Research Council. M.B.A. thanks the Wellcome Trust for personal funding.


    Footnotes
 
* Corresponding author. Tel: +44-117-9287439; Fax: +44-117-9288274; E-mail: Matthewb.Avison{at}bris.ac.uk Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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