Characterization of the role of LtgB, a putative lytic transglycosylase in Neisseria gonorrhoeae

Petra L. Kohler, Karen A. Cloud, Kathleen T. Hackett, Eric T. Beck and Joseph P. Dillard

Department of Medical Microbiology and Immunology, University of Wisconsin-Madison Medical School, Madison, 1300 University Avenue, 471A MSC, WI 53706, USA

Correspondence
Joseph P. Dillard
jpdillard{at}wisc.edu


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Neisseria gonorrhoeae releases monomeric peptidoglycan (PG) fragments during growth. These PG fragments affect pathogenesis-related phenotypes including induction of inflammatory cytokines and killing of ciliated fallopian tube cells. Although the biological activities of these molecules have been established in multiple systems, the genes and gene products responsible for their production in N. gonorrhoeae have not been determined. The authors previously identified genes for three lytic transglycosylase homologues (ltgA, ltgB and ltgC) in the N. gonorrhoeae genome sequence. Mutation of ltgA was found to affect PG fragment release, and mutation of ltgC affected cell separation. In this study the effects of complete deletion or point mutations in ltgB were characterized. Point mutations were introduced by a combination of insertion-duplication mutagenesis and positive and negative selection, thereby generating selectable marker-less mutations. The ltgB deletion mutant had normal growth characteristics and was not affected in PG fragment release. When expressed in Escherichia coli, gonococcal LtgB was able to substitute for lambda endolysin to cause cell lysis. Mutation of the predicted catalytic-site glutamic acid residue did not decrease lysis in this system. However, mutation of a nearby glutamic acid residue eliminated lysis activity.


Abbreviations: PG, peptidoglycan


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Most bacteria encode multiple lytic peptidoglycan (PG) transglycosylases. Although the function of several of these enzymes produced by Escherichia coli or bacteriophages has been characterized biochemically (reviewed by Höltje, 1998), the importance of this reaction to the bacterial cell and the need for multiple enzymes remain unclear. Lytic transglycosylases cleave the N-acetylmuramic acid–{beta}-1,4-N-acetylglucosamine linkage in PG and catalyse the formation of a 1,6-anhydro bond on the N-acetylmuramic acid (Höltje et al., 1975). Ostensibly, the biological function of these enzymes is to remove PG strands to allow for cell wall expansion during growth and for remodelling during cell division. However, E. coli mutants lacking several of the lytic transglycosylases are not affected in growth or division, and growth phenotypes are only seen in the presence of cell wall synthesis inhibitors or in strains containing mutations affecting PG-synthesizing enzymes (Lommatzsch et al., 1997). Recently, an E. coli mutant lacking all six lytic transglycosylases was shown to be slightly affected in cell separation and grew in short chains (Heidrich et al., 2002).

Liberated PG fragments contribute to virulence in infections caused by the bacterium Neisseria gonorrhoeae. Unlike most other Gram-negative bacteria, gonococci release soluble PG fragments into the surrounding milieu during growth. The major fragments released are the 1,6-anhydrodisaccharide PG monomers, the type of fragments produced by lytic transglycosylases (Sinha & Rosenthal, 1980). These PG fragments have been shown to cause the sloughing of ciliated fallopian tube cells in the organ culture model of gonococcal pelvic inflammatory disease (PID) (Melly et al., 1984). PG fragments have also been shown to induce arthritis in a rat model, producing symptoms similar to the arthritis seen in patients with disseminated gonococcal infection (DGI) (Fleming et al., 1986). PG monomers have been shown to induce production of the inflammatory cytokines IL-1 and IL-6 in human monocytes (Dokter et al., 1994). Similar PG fragments are also involved in the pathogenesis of infections caused by Bordetella pertussis and Haemophilus influenzae. In B. pertussis, released monomeric PG fragments kill ciliated cells of the trachea (Luker et al., 1995). In a rabbit model of H. influenzae meningitis, various soluble PG fragments were shown to contribute to brain oedema and leukocytosis (Burroughs et al., 1993). These results suggest that PG fragments are involved in the pathogenesis of PID and DGI, and possibly the large inflammatory response characteristic of symptomatic, uncomplicated gonorrhoea.

Although the effects of PG fragments on biological systems have been well characterized, the genes involved in production and release of these fragments have not been identified in any pathogen known to produce them. It might be hypothesized that the lytic PG transglycosylases generate the fragments, but it is unclear why the fragments are not efficiently recycled as occurs in E. coli. (For a review of PG recycling, see Park, 1995.) Do gonococci simply produce more PG monomers than can be handled by the recycling apparatus, or does production occur in some manner that favours release rather than uptake? To better understand the production and release of PG fragments by N. gonorrhoeae, we have produced mutations in lytic transglycosylase genes. These mutations will allow us to assess the role and relative contribution of each gene product to PG monomer production. Furthermore, a strain lacking lytic transglycosylase activity will allow us to directly address the contribution of released PG monomers to pathogenesis. Previously, we described the mutagenesis of ltgA and showed that mutants were partially defective in PG monomer production (Cloud & Dillard, 2002). Here we demonstrate that the putative lytic transglycosylase LtgB acts to degrade PG when produced in E. coli and can functionally replace the lytic transglycosylase of bacteriophage lambda. In addition, we describe mutations in ltgB and their effects on PG fragment release in N. gonorrhoeae.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and growth conditions.
Bacterial strains and plasmids used in this study are listed in Table 1. All experiments except transformations were performed with nonpiliated variants. Gonococci were grown with aeration in GC base liquid (GCBL) medium (1·5 % proteose peptone no. 3, 0·4 % K2HPO4, 0·1 % KH2PO4, 0·1 % NaCl; pH 7·2) containing Kellogg's supplements and 0·042 % NaHCO3, or on GCB agar plates (Difco) in 5 % CO2 at 37 °C (Kellogg et al., 1963; Morse & Bartenstein, 1974). E. coli was grown in Luria broth or on Luria agar plates (Sambrook et al., 1989). Antibiotics were used at the following concentrations: for N. gonorrhoeae, 10 µg erythromycin (Erm) ml–1 and 100 µg streptomycin (Str) ml–1; and for E. coli, 500 µg Erm ml–1 and 100 µg ampicillin (Amp) ml–1.


View this table:
[in this window]
[in a new window]
 
Table 1. Strains and plasmids

 
Cloning and mutagenesis of ltgB.
For cloning of ltgB, the following specific primers were designed based on the Gonococcal Genome Project sequence: ltgBF (5'-GACGACGGATAACCGGTGAA-3') and ltgBR (5'-TCAGAATTCGCCGTATTTCGGTTGC-3'). ltgB was amplified by PCR from MS11 chromosomal DNA (Tm 59 °C). The PCR product was digested with SmaI and EcoRI and ligated with T4 DNA ligase into pKC1. The ligation reaction was transformed into chemically competent E. coli (Active Motif, strain TAM1) following the manufacturer's instructions, and ErmR transformants were screened for a plasmid of the expected 3·5 kb size (pEB33). To construct a plasmid encoding LtgBE117A, pEB33 was amplified by PCR using mutagenic primers 5'-AAGTGGCTAGCGCGTTCCGGCAG-3' and 5'-AACGCGCTAGCCACTTCAATCAG-3'. The PCR product was gel-purified (GeneClean, Bio 101), digested with NheI, ligated, and transformed into E. coli. The resulting plasmid pEB4 included an NheI site at the site of the mutation. Plasmid construction was verified by restriction digest and DNA sequencing. Plasmid clones were sequenced at the DNA Sequence Laboratory of the University of Wisconsin Biotechnology Center using the BigDye fluorescent method as described by the manufacturer (Perkin-Elmer).

The plasmid encoding LtgBE115N (pPK44) was constructed by PCR amplifying pEB33 with mutagenic primers ltgBEtoN1 (5'-GCGATTAATCAGCCCCAACACAATCTGCGTATC-3') and ltgBEtoN2 (5'-GCGATTAATGTGGAAAGCGCGTTCCGGCAGTAT-3') (Tm 63·5 °C, extension time 1·5 min). An AseI site was introduced at the site of the mutation. The resulting PCR product was digested with AseI and self-ligated using T4 DNA ligase. The ligation reaction was transformed into competent E. coli (TAM1). ErmR isolates were screened by PCR and digestion with AseI for the mutation. Isolation of a strain containing the expected plasmid was confirmed by restriction mapping.

A plasmid for the deletion of ltgB was created by PCR amplifying ltgB and surrounding sequence with primers ltgB1 (5'-CGGCTGCTGGACGGCATTAT-3') and ltgB2 (5'-GCGGAACCATCACCACCAAG-3') (Tm 64 °C, extension time 1·5 min). The PCR product was digested with NruI and HindIII and ligated to SmaI- and HindIII-digested pKC1 to create pKH58. The coding region of ltgB was removed by PCR amplification of the plasmid using primers ltgBmut1 (5'-CGAGTGTCATGATTTTGAACCCGTGCCG-3') and ltgBmut2 (5'-AGTCAGTCATGACATAAGGAACGGGGGTGCGTC-3') (Tm 63 °C, extension time 3 min). The resulting PCR product was digested with BspHI and religated to form pKH60. The coding region was thus replaced by the sequence 5'-ATGTCATGA-3', only encoding a methionine and a serine.

ltgB mutants of N. gonorrhoeae.
N. gonorrhoeae strain MS11 (Segal et al., 1985) was transformed with pPK44 by the method of Gunn & Stein (1996) and transformants were selected on GCB-Erm plates. ErmR transformants were grown in GCBL with no antibiotic selection for 3 h and then plated on GCB-Str to select for the loss of the originally inserted plasmid. StrR colonies were transferred to GCB-Erm plates to verify loss of the plasmid from the chromosome. Presence of the mutation in ltgB was determined by PCR amplification of ltgB from mutant (PK106) chromosomal DNA and digestion with AseI. A deletion in ltgB was introduced into N. gonorrhoeae by transformation of strain MS11 with pKH60. Transformants were screened for the ltgB deletion by PCR using primers ltgB1 (5'-CGGCTGCTGGACGGCATTAT-3') and ltgB2 (5'-GCGGAACCATCACCACCAAG-3'). The resultant ltgB deletion strain was designated KH539.

Construction of strains for lysis assay.
The bacteriophage lambda lysis genes S, R and Rz were PCR amplified from lambda lysogen RLG4065 using primers lysisF (5'-GCGAATTCCCAAACTGAGCCGTAGCCAC-3') and lambdalysisR (5'-GGGTCTAGACGTAAATGGATTGCTCGG-3') (Tm 55·0 °C, extension time 1 min). The resulting PCR product and the vector, pBAD30 (Guzman et al., 1995), were digested with XbaI and EcoRI and ligated with T4 DNA ligase. Chemically competent E. coli were transformed with the ligation reaction. Transformants were grown overnight and AmpR colonies were screened for a plasmid of the expected size, 6·5 kb.

ltgB was PCR amplified directly from N. gonorrhoeae strain MS11 colonies with primers ltgBlysisF (5'-CGGGTACCTTATGAAAAAACCGACCGATACC-3') and ltgBlysisR (5'-CGGAGCTCTCAGCGCCACTGCCAGCG-3') (Tm 60·0 °C, extension time 1 min). In order to create a vector with the lambda genes S and Rz but not R, pPK20 was PCR amplified from the end of S upstream and from the beginning of Rz downstream using the primers SacIlysisrevF (5'-CGTGCGAGCTCATGAGCAGAGTCACCGCG-3') and lysisreverseR (5'-CGCGGTACCTTATTGATTTCTACCATCTTCTACTCC-3') (Tm 60·0 °C, extension time 3 min). The resulting PCR products were digested with SacI and KpnI and ligated using T4 DNA ligase. The ligation reaction was transformed into E. coli as described above and AmpR colonies were screened for a plasmid of the expected size of pPK33. The plasmid was mapped by restriction digestion to confirm the insertion of ltgB. The plasmid pPK46, encoding S, Rz and LtgBE115N, was constructed using the same method as described for constructing pPK33 except that ltgB encoding the point mutation was PCR amplified from pPK44. The plasmid pPK39, encoding S, Rz and LtgBE117A, was also constructed using the same method as used to construct pPK33 except that ltgB encoding the point mutation was PCR amplified from pEB4.

Lysis assay.
Strains were grown overnight in LB broth with Amp and then diluted to an OD550 of 0·6 in M9 minimal medium with glycerol and Amp. Cultures were induced with 0·2 % L-arabinose and grown at 37 °C with aeration. The OD550 was measured immediately upon induction and every 30 min thereafter for 300 min using a Beckman DU-64 spectrophotometer.

Peptidoglycan assays.
PG purification methods and PG turnover assays were performed essentially as described by Cloud & Dillard (2002). For PG fragment release assays, PG was pulse-labelled with 2 µCi ml–1 (74 kBq ml–1) [6-3H]glucosamine (Amersham) in GCBL containing pyruvate as the carbon source. For each time point, the cultures were centrifuged, and the macromolecular PG was harvested using the boiling SDS method. Turnover was measured as loss of radioactivity from the macromolecular PG fraction over time. For characterization of released PG fragments, supernatants from exponential-phase cultures were applied to size-exclusion columns and fractions were assayed for radioactivity by scintillation counting. Statistical significance was determined by Student's t test.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
We previously described the identification of three lytic transglycosylase homologues in the gonococcal genome sequence. The first gene, designated ltgA, was characterized and found to be involved in PG monomer production (Cloud & Dillard, 2002). The second gene, designated ltgB, is characterized here. We recently showed that the third gene, ltgC, is involved in cell separation (Cloud & Dillard, 2004).

ltgB encodes a homologue of E. coli MltC. Although the effects of single mltC mutations have not been reported, purified E. coli MltC has been shown to have lytic transglycosylase activity in vitro (Dijkstra & Keck, 1996). LtgB is 31 % identical and 47 % similar to MltC over a 100 amino acid region, and the sequence motifs common to family 1 lytic transglycosylases are conserved in LtgB (Fig. 1) (Blackburn & Clarke, 2001). One difference between LtgB and MltC is that the LtgB predicted amino acid sequence does not contain a lipoprotein-processing site, and thus LtgB is not likely to be a membrane-bound lipoprotein as is MltC. In addition to the similarity to MltC, LtgB also shares over 90 % amino acid identity with putative lytic transglycosylases in the two sequenced strains of Neisseria meningitidis. Examination of the N. gonorrhoeae genome sequence in the ltgB region shows that just upstream of ltgB is a copy of the insertion sequence IS1106, and immediately downstream of ltgB is a gene for a hypothetical transporter of the major facilitator family.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 1. Multiple sequence alignment of N. gonorrhoeae LtgB with E. coli MltC and Slt70. Only a portion of each protein sequence is shown. Amino acids in bold are conserved in all three proteins. Motif boundaries for the family 1 lytic transglycosylases, as defined by Blackburn & Clarke (2001), are indicated below the alignment. Residues marked with a star were changed by mutation; the black star indicates loss of PG degradation activity.

 
LtgB can functionally replace the lytic transglycosylase in the bacteriophage lambda lysis system
The homology of LtgB with MltC suggests that LtgB is a PG lytic transglycosylase. We investigated the ability of LtgB to function in PG degradation by using LtgB to replace the PG transglycosylase in the bacteriophage lambda lysis system. Lambda encodes three gene products necessary for lysis of the host cell during the lytic stage of the bacteriophage lifecycle, R, S and Rz (Young, 1992). R is the PG transglycosylase necessary for degradation of the cell wall during lysis (Bienkowska-Szewczyk et al., 1981). S is a holin, a small transmembrane protein necessary to allow entry of R into the periplasm (Young, 1992). The function of Rz is unclear, although the locus encodes two gene products, Rz and Rz1 (Hanych et al., 1993). We cloned S, R and Rz from lambda and placed them under the control of an arabinose-inducible promoter to create the plasmid pPK20. Upon induction with arabinose, E. coli carrying pPK20 lysed rapidly (Fig. 2). We next constructed a plasmid containing ltgB instead of R in order to determine whether LtgB could functionally replace R in the lambda lysis system. The strain of E. coli carrying the plasmid containing S, ltgB and Rz (pPK33) lysed when arabinose was added to the culture medium, though not as quickly as the strain carrying the plasmid encoding the lambda lysis genes, pPK20 (Fig. 2). This result suggests that LtgB degrades PG and may function as an autolysin.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 2. Induced lysis of E. coli. An arabinose-inducible promoter was used to drive expression of the bacteriophage lambda genes necessary for lysis. The gene encoding the lytic transglycosylase from lambda (R) was replaced with ltgB and ltgB point mutants. Expression of ltgB compensates for the loss of R, and a point mutation at glutamic acid 115 eliminates enzymic activity of LtgB. The values are the means from three separate experiments, and the error bars indicate the standard error of the mean.

 
The similarity of LtgB to MltC suggests that LtgB, like MltC, is a PG transglycosylase, i.e. it is predicted to cleave the N-acetylmuramic acid–{beta}-1,4-N-acetylglucosamine bond and create a 1,6-anhydro bond on the N-acetylmuramic acid (Dijkstra & Keck, 1996). Residues important in this reaction have been characterized in the E. coli lytic transglycosylases Slt70 and Slt35 and in the lambda lytic transglycosylase R (Jespers et al., 1992; Leung et al., 2001; Thunnissen et al., 1994, 1995; van Asselt et al., 1999a, b, 2000). In order to examine the function of LtgB as a putative PG transglycosylase we made a point mutation in the putative active site of LtgB. The PG transglycosylases contain a conserved acidic residue necessary for enzymic activity. The family 1 PG transglycosylases, which include MltC, contain an invariant glutamic acid positioned adjacent to an invariant serine (Blackburn & Clarke, 2001). We identified a putative catalytic glutamic acid in LtgB by alignment with MltC (Fig. 1). A point mutation was made at this residue, changing glutamic acid 117 to alanine (LtgBE117A). ltgBE117A was substituted for R in pPK20, resulting in pPK36. Surprisingly, when E. coli cells carrying pPK36 were induced with arabinose they displayed lysis, though lysis was not as rapid as seen with pPK33 carrying the wild-type ltgB (Fig. 2).

The amino acid sequence of LtgB revealed another glutamic acid at position 115, very close to the predicted catalytic residue, E117 (Fig. 1). Because glutamic acid 115 is only three residues away from the conserved serine, it was a candidate for the essential glutamic acid residue necessary for PG transglycosylase activity. We substituted R in pPK20 with a version of ltgB containing a point mutation altering glutamic acid 115 to asparagine (ltgBE115N). E. coli carrying the resulting plasmid, pPK46, did not lyse upon induction with arabinose (Fig. 2). A second ES motif is present at residues 99–100 of the putative LtgB amino acid sequence. Therefore we also made a point mutation at glutamic acid 99 to determine if E99 might be the necessary glutamic acid for PG transglycosylase activity. When the gene encoding this mutated version of LtgB (ltgBE99A) was substituted for R in the lambda lysis system (pPK39), lysis of E. coli was observed upon induction with arabinose (data not shown). These results suggest that glutamic acid 115 is necessary for the PG degradation activity of LtgB, and bring into question whether LtgB truly functions as a PG transglycosylase or may function as a peptidoglycanase with a different specificity.

Creation of a point mutation in ltgB in the gonococcal chromosome using an insertional plasmid and positive and negative selection
We have sought to create marker-less mutations in the lytic transglycosylase genes as we are attempting to make gonococcal mutants with multiple disruptions. In our mutagenesis of ltgA, we used a cassette containing ermC, an Erm resistance marker, and rpsL, a Str sensitivity marker, to interrupt ltgA in the gonococcal chromosome (Cloud & Dillard, 2002). The insertion was subsequently replaced in a second transformation using a plasmid carrying an in vitro-constructed deletion, a method developed for gonococci by Johnston & Cannon (1999). We devised a shortened method for introduction of a point mutation into ltgB (Fig. 3). A plasmid containing ltgBE115N was constructed using pKC1, the plasmid carrying ermC and rpsL, as the parent vector. The resulting plasmid, pPK44, was transformed into N. gonorrhoeae MS11, and ErmR StrS transformants were obtained. Strain MS11, like many other gonococcal strains, is naturally StrR. However, Str sensitivity is dominant over Str resistance when both alleles of the gene are expressed (Lederberg, 1951). In order to select for isolates that had lost the inserted plasmid, ErmR transformants were grown without antibiotics in liquid culture and plated to medium containing Str. These isolates were replica-plated to medium containing Str or Erm to confirm Str resistance and to test for Erm sensitivity. Of 20 StrR ErmS isolates screened for the mutation by PCR and restriction digestion, 18 had retained the point mutation. The resulting gonococcal strain encoding LtgBE115N was designated PK106.



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 3. Schematic of insertion-duplication, positive/negative selection mutagenesis. Plasmids containing ermC (Erm resistance), rpsL (Str sensitivity) and ltgB with the mutation of interest were transformed into MS11, and ErmR colonies selected. ErmR transformants were transferred to medium containing Str. StrR colonies were screened for mutations in ltgB (represented by a star) by PCR and restriction endonuclease digestion.

 
Several elements of the mutagenesis were surprising. The frequency of StrR isolates was much higher than expected for resolution resulting in loss of the plasmid from the chromosome. In our previous experiments with inserted plasmids, we were unable to detect loss of the insertion in 105 cells (Hamilton et al., 2001). However, in the ltgB mutagenesis, StrR colonies were obtained at a frequency of 8·1x10–2±2x10–2. Also, Str resistance did not always indicate loss of the inserted plasmid. When StrR colonies were plated to medium containing Erm, 11·5 % of StrR colonies were also ErmR, indicating that the original plasmid we had introduced, pPK44, was still inserted in the chromosome. To further investigate the appearance of StrR ErmR gonococci, we PCR amplified and sequenced rpsL from the inserted plasmid in four StrR ErmR isolates. Three of these isolates contained the same point mutation as is found in the original chromosomal copy of rpsL. The fourth contained a nonsense mutation in rpsL.

Characterization of ltgB mutants of N. gonorrhoeae
In order to examine the function of ltgB in N. gonorrhoeae, an in-frame deletion was created in ltgB. A plasmid containing the region surrounding ltgB and an in-frame deletion of the ltgB coding sequence was used to transform wild-type gonococcal strain MS11. We screened by PCR for homologous recombination and the loss of ltgB. The strain carrying the deletion of ltgB was designated KH539. We characterized this strain and PK106, carrying the ltgBE115N mutation, for effects on PG fragment release.

To characterize PG turnover, gonococcal strains were grown in medium containing [6-3H]glucosamine and lacking glucose, to metabolically label the PG. Macromolecular PG was collected during a chase period and the amount of radioactivity present was determined to quantify the amount of the original PG that remained in the cell wall and the rate at which fragments were released into the culture supernatant. If LtgB contributes to PG fragment release, then the ltgB mutants would be expected to show a reduced rate of PG turnover. However, no significant difference in PG turnover was observed between the wild-type and mutant strains (Fig. 4).



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 4. Mutation of ltgB does not affect PG turnover. MS11 (wild-type), KH539 (ltgB deletion), and PK106 (ltgBE117N) were each pulse-labelled with [6-3H]glucosamine and grown in liquid culture. Aliquots were taken, the macromolecular PG harvested, and the 3H content measured at different times. The values shown are the arithmetic means of the percentages of original radioactivity remaining from at least three trials. Error bars indicate the standard error of the mean.

 
To determine whether the same types of soluble PG fragments were being released during growth of the mutant and wild-type strains, the PG fragments in culture supernatants from KH539 and MS11 were analysed by size-exclusion chromatography. KH539 exhibited a fragment profile similar to that of the wild-type strain (Fig. 5). The amounts of PG multimers, PG monomers and free disaccharide released by the ltgB mutant were not different from those of the wild-type. This result indicates that ltgB is not involved in the release of cytotoxic PG monomers by gonococci.



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 5. An ltgB mutant is able to release PG monomers as well as wild-type gonococci. Supernatants from each strain, containing 3H-labelled PG fragments, were separated by passage over gel-filtration columns. Fractions were collected and their radioactivity measured. Data are presented as percentage of counts per minute in the included volume versus the volume passed through the columns. PG fragments were identified by comparison with chromatographs of known standards.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The data presented here indicate that LtgB functions in PG degradation. LtgB was able to function in place of the lytic transglycosylase of the bacteriophage lambda lysis system. This activity and the sequence similarity with known lytic transglycosylases suggest that LtgB is a lytic transglycosylase, but further characterization of its biochemical activity would be necessary to demonstrate this function conclusively. Unfortunately, expression of ltgB in E. coli resulted in insoluble protein, and we were not able to assay the biochemical function of LtgB directly. Sequence analysis has shown that a glutamic acid directly adjacent to a serine is conserved among lytic transglycosylases (Blackburn & Clarke, 2001; Dijkstra & Thunnissen, 1994; Koonin & Rudd, 1994). This conserved glutamic acid is hypothesized to be the catalytic residue in lytic transglycosylases as it has been demonstrated to be for Slt70, Slt35 and lambda R (Jespers et al., 1992; Leung et al., 2001; Thunnissen et al., 1994, 1995; van Asselt et al., 1999a, b, 2000). The predicted amino acid sequence of LtgB shows a conserved glutamic acid residue at amino acid 117 adjacent to a serine. This group of residues aligns well with the predicted catalytic site of MltC from E. coli (Fig. 1) and the catalytic glutamic acid of E. coli Slt70. However, mutation of the putative active-site glutamic acid (E117) did not attenuate the enzymic activity of LtgB as measured by activity in the lambda lysis system. Further mutational analysis revealed that glutamic acid 115 (E115) is essential for LtgB function because a mutation changing E115 to an asparagine eliminated the ability of LtgB to lyse E. coli in the lambda lysis system. It is unclear how mutation of E115 might affect the function of LtgB. Replacement of E115 in LtgB with asparagine may not allow for proper conformation for substrate binding. Examination of the alignment of LtgB and MltC from E. coli (Fig. 1) reveals that in MltC there is a glutamine residue in the position occupied by E115 in LtgB. Thus, mutation of LtgB at E115 makes LtgBE115N more similar to MltC at position 115 (position 216 in MltC) than wild-type LtgB. It is possible that E115 is the catalytic residue and, even though not in the expected position in the primary sequence, it may be in the correct position in the secondary structure to carry out the lytic transglycosylase reaction. Alternatively, this result may indicate that LtgB cleaves PG at a different bond from lytic transglycosylases and may have a different specificity from the related E. coli enzymes. Database searches using LtgB as the query showed that there are at least 17 more examples of lytic transglycosylase homologues with the ExES pattern that we observe in LtgB.

The studies presented here demonstrate the utility of combining insertion-duplication mutagenesis and positive and negative selection for the introduction of mutations into the gonococcal chromosome. Mutants were obtained at an appreciable frequency, and the undesired isolates that became Str sensitive but did not resolve out the duplication could be screened out by replica plating on medium containing Str and Erm. The reason for the high rate of StrR ErmR isolates is not completely clear, but we found two different types of mutations in the introduced rpsL that resulted in StrR. Since three of the four mutations we found were identical to the sequence in the original StrR allele of rpsL, it appears that recombination between the resident rpsL (StrR) and the rpsL (StrS) on the inserted plasmid resulted in these mutations through a gene-conversion event. However, we also found a nonsense mutation in one of the StrR ErmR isolates. This result, together with the high frequency of ErmR StrR isolates, suggests that the production of both RpsL proteins may be detrimental. A similar phenomenon was observed when rpsL was used as a counterselectable marker in Streptococcus pneumoniae (Sung et al., 2001).

Examination of PG turnover in the ltgB deletion mutant shows that LtgB does not significantly affect the amount of PG released. In addition, the ltgB deletion mutant still produced PG monomers in amounts no different from the wild-type. Thus it is clear that LtgB does not substantially contribute to PG monomer production. This result raises the question of what the function of LtgB may be in N. gonorrhoeae. It is clear from E. coli lysis assays that LtgB can degrade PG. Thus it might function in the removal of PG strands during growth or division, or for breaking down fragments for PG recycling. However, we did not observe any defects in growth or division in the ltgB mutants, and we would expect mutants defective in recycling to show higher rates of PG turnover. A lack of phenotypes is also observed for E. coli lytic transglycosylase mutants. A triple lytic transglycosylase mutant in E. coli did not show any growth phenotypes (Lommatzsch et al., 1997), and a mutant lacking six lytic transglycosylases was only slightly affected in cell separation (Heidrich et al., 2002). It has been proposed that there is functional redundancy in PG hydrolases in E. coli, i.e. one of the many other PG-degrading enzymes may substitute for the lost function (Heidrich et al., 2002). This could be the case in N. gonorrhoeae as well. The common N. gonorrhoeae chromosome encodes five lytic transglycosylase homologues (Blackburn & Clarke, 2001; Cloud & Dillard, 2002, 2004) (GenBank accession AE004969), and the gonococcal genetic island present in most gonococcal strains encodes two additional lytic transglycosylase homologues (Dillard & Seifert, 1997; Hamilton et al., 2005). One of these enzymes or the PG-degrading amidase, endopeptidase or glucosaminidase (Chapman & Perkins, 1983; Hebeler & Young, 1976; Stefanova et al., 2003) of gonococci may substitute for LtgB function.


   ACKNOWLEDGEMENTS
 
This work was supported by NIH grant AI47958 to J. P. D. P. L. K was supported by NIH grant T32 GM007215. We thank R. S. Rosenthal for the kind gift of PG fragment standards. We acknowledge the Gonococcal Genome Sequencing Project supported by USPHS/NIH grant AI38399 and B. A. Roe, L. Song, S. P. Lin, X. Yuan, S. Clifton, T. Ducey, L. Lewis and D. W. Dyer of the University of Oklahoma. We thank K. T. Forest for helpful discussions and critical reading of the manuscript, and T. Gaal for providing lambda lysogen RLG4065.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bienkowska-Szewczyk, K., Lipinska, B. & Taylor, A. (1981). The R gene product of bacteriophage lambda is the murein transglycosylase. Mol Gen Genet 184, 111–114.[CrossRef][Medline]

Blackburn, N. T. & Clarke, A. J. (2001). Identification of four families of peptidoglycan lytic transglycosylases. J Mol Evol 52, 78–84.[Medline]

Burroughs, M., Prasad, S., Cabellos, C., Mendelman, P. M. & Tuomanen, E. (1993). The biological activities of peptidoglycan in experimental Haemophilus influenzae meningitis. J Infect Dis 167, 464–468.[Medline]

Chapman, S. J. & Perkins, H. R. (1983). Peptidoglycan-degrading enzymes in ether-treated cells of Neisseria gonorrhoeae. J Gen Microbiol 129, 877–883.[Medline]

Cloud, K. A. & Dillard, J. P. (2002). A lytic transglycosylase of Neisseria gonorrhoeae is involved in peptidoglycan-derived cytotoxin production. Infect Immun 70, 2752–2757.[Abstract/Free Full Text]

Cloud, K. A. & Dillard, J. P. (2004). Mutation of a single lytic transglycosylase causes aberrant septation and inhibits cell separation of Neisseria gonorrhoeae. J Bacteriol 186, 7811–7814.[Abstract/Free Full Text]

Dijkstra, A. & Keck, W. (1996). Identification of new members of the lytic transglycosylase family in Haemophilus influenzae and Escherichia coli. Microb Drug Resist 2, 141–145.[Medline]

Dijkstra, B. W. & Thunnissen, A.-M. W. H. (1994). ‘Holy’ proteins. II. The soluble lytic transglycosylase. Curr Opin Struct Biol 4, 810–813.[CrossRef][Medline]

Dillard, J. P. & Seifert, H. S. (1997). A peptidoglycan hydrolase similar to bacteriophage endolysins acts as an autolysin in Neisseria gonorrhoeae. Mol Microbiol 25, 893–901.[CrossRef][Medline]

Dokter, W. H. A., Dijkstra, A. J., Koopmans, S. B., Stulp, B. K., Keck, W., Halie, M. R. & Vellenga, E. (1994). G(Anh)MTetra, a natural bacterial cell wall breakdown product induces interleukin-1beta and interleukin-6 expression in human monocytes. J Biol Chem 269, 4201–4206.[Abstract/Free Full Text]

Fleming, T. J., Wallsmith, D. E. & Rosenthal, R. S. (1986). Arthropathic properties of gonococcal peptidoglycan fragments: implications for the pathogenesis of disseminated gonococcal disease. Infect Immun 52, 600–608.[Medline]

Gunn, J. S. & Stein, D. C. (1996). Use of a nonselective transformation technique to construct a multiply restriction/modification-deficient mutant of Neisseria gonorrhoeae. Mol Gen Genet 251, 509–517.[CrossRef][Medline]

Guzman, L. M., Belin, D., Carson, M. J. & Beckwith, J. (1995). Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177, 4121–4130.[Abstract/Free Full Text]

Hamilton, H. L., Schwartz, K. J. & Dillard, J. P. (2001). Insertion-duplication mutagenesis of Neisseria: use in characterization of DNA transfer genes in the gonococcal genetic island. J Bacteriol 183, 4718–4726.[Abstract/Free Full Text]

Hamilton, H. L., Domínguez, N. M., Schwartz, K. J., Hackett, K. T. & Dillard, J. P. (2005). Neisseria gonorrhoeae secretes chromosomal DNA via a novel type IV secretion system. Mol Microbiol 55, 1704–1721.[CrossRef][Medline]

Hanych, B., Kedzierska, S., Walderich, B., Uznanski, B. & Taylor, A. (1993). Expression of the Rz gene and the overlapping Rz1 reading frame present at the right end of the bacteriophage lambda genome. Gene 129, 1–8.[CrossRef][Medline]

Hebeler, B. H. & Young, F. E. (1976). Mechanism of autolysis of Neisseria gonorrhoeae. J Bacteriol 126, 1186–1193.[Medline]

Heidrich, C., Ursinus, A., Berger, J., Schwarz, H. & Höltje, J.-V. (2002). Effects of multiple deletions of murein hydrolases on viability, septum cleavage, and sensitivity to large toxic molecules in Escherichia coli. J Bacteriol 184, 6093–6099.[Abstract/Free Full Text]

Höltje, J.-V. (1998). Growth of the stress-bearing and shape-maintaining murein sacculus of Escherichia coli. Microbiol Mol Biol Rev 62, 181–203.[Abstract/Free Full Text]

Höltje, J.-V., Mirelman, D., Sharon, N. & Schwartz, U. (1975). Novel type of murein transglycosylase in Escherichia coli. J Bacteriol 124, 1067–1076.[Medline]

Jespers, L., Sonveaux, E. & Fastrez, J. (1992). Is the bacteriophage lambda lysozyme an evolutionary link or a hybrid between the C and V-type lysozymes? Homology analysis and detection of the catalytic amino acid residues. J Mol Biol 228, 529–538.[CrossRef][Medline]

Johnston, D. M. & Cannon, J. G. (1999). Construction of mutant strains of Neisseria gonorrhoeae lacking new antibiotic markers using a two gene cassette with positive and negative selection. Gene 236, 179–184.[CrossRef][Medline]

Kellogg, D. S., Jr, Peacock, W. L., Jr, Deacon, W. E., Brown, L. & Pirkle, C. L. (1963). Neisseria gonorrhoeae. I. Virulence genetically linked to clonal variation. J Bacteriol 85, 1274–1279.[Medline]

Koonin, E. V. & Rudd, K. E. (1994). A conserved domain in putative bacterial and bacteriophage transglycosylases. Trends Biochem Sci 19, 106–107.[CrossRef][Medline]

Lederberg, J. (1951). Streptomycin resistance: a genetically recessive mutation. J Bacteriol 61, 549–550.[Medline]

Leung, A. K., Duewel, H. S., Honek, J. F. & Berghuis, A. M. (2001). Crystal structure of the lytic transglycosylase from bacteriophage lambda in complex with hexa-N-acetylchitohexaose. Biochemistry 40, 5665–5673.[CrossRef][Medline]

Lommatzsch, J., Templin, M. F., Kraft, A. R., Vollmer, W. & Höltje, J.-V. (1997). Outer membrane localization of murein hydrolases: MltA, a third lipoprotein lytic transglycosylase in Escherichia coli. J Bacteriol 179, 5465–5470.[Abstract/Free Full Text]

Luker, K. E., Tyler, A. N., Marshall, G. R. & Goldman, W. E. (1995). Tracheal cytotoxin structural requirements for respiratory epithelial damage in pertussis. Mol Microbiol 16, 733–743.[Medline]

Melly, M. A., McGee, Z. A. & Rosenthal, R. S. (1984). Ability of monomeric peptidoglycan fragments from Neisseria gonorrhoeae to damage human fallopian-tube mucosa. J Infect Dis 149, 378–386.[Medline]

Morse, S. A. & Bartenstein, L. (1974). Factors affecting autolysis of Neisseria gonorrhoeae. Proc Soc Exp Biol Med 145, 1418–1421.

Park, J. T. (1995). Why does Escherichia coli recycle its cell wall peptides? Mol Microbiol 17, 421–426.[Medline]

Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Segal, E., Billyard, E., So, M., Storzbach, S. & Meyer, T. F. (1985). Role of chromosomal rearrangement in N. gonorrhoeae pilus phase variation. Cell 40, 293–300.[CrossRef][Medline]

Sinha, R. K. & Rosenthal, R. S. (1980). Release of soluble peptidoglycan from growing gonococci: demonstration of anhydro-muramyl-containing fragments. Infect Immun 29, 914–925.[Medline]

Stefanova, M. E., Tomberg, J., Olesky, M., Höltje, J.-V., Gutheil, W. G. & Nicholas, R. A. (2003). Neisseria gonorrhoeae penicillin-binding protein 3 exhibits exceptionally high carboxypeptidase and beta-lactam binding activities. Biochemistry 42, 14614–14625.[CrossRef][Medline]

Sung, C. K., Li, H., Claverys, J. P. & Morrison, D. A. (2001). An rpsL cassette, Janus, for gene replacement through negative selection in Streptococcus pneumoniae. Appl Environ Microbiol 67, 5190–5196.[Abstract/Free Full Text]

Thunnissen, A.-M. W. H., Dijkstra, A. J., Kalk, K. H., Rozeboom, H. J., Engel, H., Keck, W. & Dijkstra, B. W. (1994). Doughnut-shaped structure of a bacterial muramidase revealed by X-ray crystallography. Nature 367, 750–753.[CrossRef][Medline]

Thunnissen, A.-M. W. H., Rozeboom, H. J., Kalk, K. H. & Dijkstra, B. W. (1995). Structure of the 70-kDa soluble lytic translgycosylase complexed with bulgecin A. Implications for the enzymatic mechanism. Biochemistry 34, 12729–12737.[CrossRef][Medline]

van Asselt, E. J., Dijkstra, A. J., Kalk, K. H., Takacs, B., Keck, W. & Dijkstra, B. W. (1999a). Crystal structure of Escherichia coli lytic transglycosylase Slt35 reveals a lysozyme-like catalytic domain with an EF-hand. Structure Fold Des 7, 1167–1180.[CrossRef][Medline]

van Asselt, E. J., Thunnissen, A.-M. W. H. & Dijkstra, B. W. (1999b). High resolution crystal structures of the Escherichia coli lytic transglycosylase Slt70 and its complex with a peptidoglycan fragment. J Mol Biol 291, 877–898.[CrossRef][Medline]

van Asselt, E. J., Kalk, K. H. & Dijkstra, B. W. (2000). Crystallographic studies of the interactions of Escherichia coli lytic transglycosylase Slt35 with peptidoglycan. Biochemistry 39, 1924–1934.[CrossRef][Medline]

Young, R. (1992). Bacteriophage lysis: mechanism and regulation. Microbiol Rev 56, 430–481.[Medline]

Received 16 April 2005; revised 25 June 2005; accepted 30 June 2005.



This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Kohler, P. L.
Articles by Dillard, J. P.
Articles citing this Article
PubMed
PubMed Citation
Articles by Kohler, P. L.
Articles by Dillard, J. P.
Agricola
Articles by Kohler, P. L.
Articles by Dillard, J. P.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
INT J SYST EVOL MICROBIOL MICROBIOLOGY J GEN VIROL
J MED MICROBIOL ALL SGM JOURNALS
Copyright © 2005 Society for General Microbiology.