A peptidoglycan hydrolase similar to bacteriophage endolysins acts as an autolysin in Neisseria gonorrhoeae

Molecular Microbiology (1997) 25(5), 893–901

A peptidoglycan hydrolase similar to bacteriophage endolysins acts as an autolysin in Neisseria gonorrhoeae Joseph P. Dillard* and H. Steven Seifert Department of Microbiology–Immunology, Northwestern University Medical School, Chicago, IL 60611, USA.

child-bearing age and often leads to infertility (Aral et al., 1991). Gonococci also infect the eyes of newborn infants, causing an infection that can lead to blindness. Untreated gonorrhoea can lead to disseminated gonococcal infection that can be manifested as arthritis, endocarditis or meningitis. Gonococci are very fastidious and quickly lose viability under multiple conditions, such as depletion of nutrients, a shift to low temperature or the presence of cell wall synthesis inhibitors or chaotropic agents (Morse and Bartenstein, 1974; Elmros et al., 1976a, b; Goodell et al., 1978). The molecular mechanisms underlying this phenomenon remain unclear. However, microscopic examinations of cells exposed to such conditions have shown lysed cells (Morse and Bartenstein, 1974; Elmros et al., 1976a; Goodell et al., 1978), suggesting that peptidoglycan-hydrolysing enzymes are involved. Peptidoglycan (PG) is composed of repeating b(1–4)linked disaccharides of N -acetylglucosamine b(1–4) linked to N -acetylmuramic acid. The muramic acid has an amide-linked peptide side-chain, and peptide bonds formed between a portion of these side-chains cross-link the glycan strands together to form a macromolecular cage that is the cell wall. Changes to the size or shape of the bacterium, which occur during growth, septation or sporulation, require the hydrolysis of PG (Holtje and Tuomanen, 1991). Some of the PG hydrolysing enzymes that act on the macromolecular cell wall are capable of lysing the cell and are thus known as autolysins. Autolysis and PG hydrolysis in N. gonorrhoeae are not only interesting from the point of view of bacterial physiology but may also be involved in genetic transformation and pathogenesis. Gonococci undergo natural transformation (Sparling, 1966), and the donor DNA is thought to be provided by lysed cells (Norlander et al., 1979; Seifert et al., 1988). Gonococci also release monomeric fragments of PG (Sinha and Rosenthal, 1980) that have multiple biological activities, including killing ciliated fallopian tube cells in the organ culture model of PID (Melly et al., 1984; Fleming et al., 1986; Krueger et al., 1987; Dokter et al., 1994). Several biochemical activities have been described that could contribute to PG hydrolysis and autolysis in gonococci. Hebeler and Young characterized a pH-dependent N -acetylmuramyl-L-alanine amidase activity and found that this was the major cell-lysing activity in vitro (Hebeler

Summary We have identified a gene encoding an autolysin (atlA) from Neisseria gonorrhoeae. The deduced amino acid sequence of AtlA shows significant similarity to the peptidoglycan degrading transglycosylases (endolysins) of bacteriophages lambda and P2, suggesting that the encoded protein also functions in peptidoglycan hydrolysis. An atlA mutant was identical to the wild-type strain in exponential growth rate, but demonstrated reduced lysis and peptidoglycan turnover in the stationary phase of growth. When transferred into a buffer solution, at a pH non-permissive for other gonococcal autolysins, an autolytic activity was detectable in the wild-type strain that was not present in the mutant. The most dramatic phenotype of the mutant occurred after extended time in stationary phase. After approximately 16 h in stationary phase, both strains underwent an apparent replication event, after which the wild-type strain died rapidly whereas the atlA mutant survived considerably longer. Even after both the wild-type and mutant cells were dead, many of the mutant cells maintained intact morphology, whereas the wild-type cells were lysed. These results suggest that AtlA is a peptidoglycan transglycosylase related to bacteriophage endolysins and acts as an autolysin in the stationary phase. Introduction

Neisseria gonorrhoeae is a Gram-negative bacterium and the causative agent of the sexually transmitted disease gonorrhoea. Gonococcal infection generally causes uncomplicated urethritis or cervicitis that is easily treatable. However, in women, N. gonorrhoeae can cause a more serious disease. N. gonorrhoeae causes a large proportion of the cases of pelvic inflammatory disease (PID), a painful and chronic condition that affects 1 out of 10 women of Received 13 May, 1997; revised 7 July, 1997; accepted 8 July, 1997. *For correspondence. E-mail [email protected]; Tel. (312) 503 9786; Fax (312) 503 1339. Q 1997 Blackwell Science Ltd

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894 J. P. Dillard and H. S. Seifert and Young, 1975; 1976). Chapman and Perkins (1983) described an endopeptidase activity in gonococcal extracts capable of cleaving the peptide cross-links present in PG (Chapman and Perkins, 1983). Gubish et al. (1982) found an N -acetylglucosaminidase activity that cleaves the glycan backbone. Sinha and Rosenthal (1980) characterized PG fragments released from growing gonococci and found that the most abundant fragment was the 1,6 anhydro PG monomer, indicating the presence of a highly active PG transglycosylase (Sinha and Rosenthal, 1980). The uncontrolled activity of any of these enzymes could be responsible for the autolysis seen in gonococcal cultures under non-growth conditions or other undescribed activities may be responsible. None of the genes for these autolysins has been identified. We have identified a gene in N. gonorrhoeae (atlA) whose deduced amino acid sequence is similar to PG transglycosylases (endolysins) from bacteriophages l and P2. The bacteriophage enzymes cause bacterial cell lysis by degrading the cell wall, thus allowing phage particles to be released (Young, 1992). The l endolysin (gene product R, gpR) is a PG transglycosylase; it degrades PG by hydrolysing the b(1–4) bond between muramic acid and N -acetylglucosamine and creates an intramolecular 1,6 anhydro bond on the muramic acid (Bienkowska-Szewczyk et al., 1981), thus creating a 1,6-anhydro-PG monomer fragment identical to that released by gonococci. Consistent with its similarity to the endolysins, AtlA appears to act in PG hydrolysis. A mutation in atlA is shown to specifically affect stationary phase death, autolysis and PG metabolism. Results

Identification of an endolysin homologue in N. gonorrhoeae Previous reports from this laboratory described the use of a mini-TnphoA for identifying genes for exported proteins in N. gonorrhoeae (Boyle-Vavra and Seifert, 1994). While engaged in DNA sequencing of the gene for one exported protein, we identified a nearby ORF showing significant

similarity to endolysins from bacteriophages lambda and P2 (Fig. 1). These endolysins are PG transglycosylases that break down the bacterial cell wall to release phage particles during the lytic phase of infection (BienkowskaSzewczyk et al., 1981; Ziermann et al., 1994). We have designated our gene atlA for autolysin A, as our data indicate that the encoded protein plays a role in cell lysis (see below).

DNA sequence analysis of the atlA region The deduced amino acid sequence of AtlA shows a significant degree of similarity to two bacteriophage PG transglycosylases, with 56% similarity, 38% identity to the l R gene product and 58% similarity, 39% identity to the P2 K gene product (Fig. 1). One obvious difference between AtlA and the bacteriophage transglycosylases is a predicted 28 amino acid extension on the N-terminus of AtlA. This region of the protein does not show homology to any known sequence and does not contain an identifiable signal sequence. In bacteriophages, endolysins lack signal sequences and instead are transported through pores formed by transmembrane proteins known as holins (Young, 1992). Genes for holins are usually found adjacent to genes for endolysins in bacteriophages. Holin proteins are small and not very similar by sequence, but have similar structural features that can be predicted based on the sequence (Young, 1992). To determine if a holin gene was present adjacent to atlA , DNA sequence from around atlA was obtained. DNA sequencing revealed the end of one large ORF directly upstream of atlA (orf1 ), and one small ORF (orf2 ) directly downstream (Fig. 2). Further downstream were two ORFs reading in the opposite direction (orf3, orf4 ). ORF4 showed significant similarity to cold shock proteins from other bacteria (data not shown). The other ORFs did not have significant similarity to any sequence in the available databases. Although ORF2 is the correct size to be a holin, it does not have the required structural features, i.e. two membrane-spanning domains separated by beta turns. ORF3 is a small protein with two predicted

Fig. 1. Amino acid alignment of AtlA with bacteriophage endolysins, lambda gpR (l R) and P2 gpK (P2 K). Identical amino acids are indicated by dark shading.

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Autolysin of N. gonorrhoeae 895

Fig. 2. Chromosomal map of the atlA region in MS11 A and JD1510. The restriction map of the atlA region of the MS11 A chromosome was created based on Southern analyses using the atlA coding region as the probe. The positions of genes are based on DNA sequence data. The maps of JD1510 and JD1511 chromosomes were similarly determined using the ermC and atlA genes as probes. The JD1511 chromosome is identical to that of 1510 except that the ermC gene is facing in the opposite direction The ermC gene is not drawn to scale. Only the Dra I sites in atlA are shown to indicate the position of the mutation in JD1510/ 1511. Restriction sites are: B, Bam HI; C, Cla I; D, Dra I; H, Hin dIII; P, Pst I. Parentheses are used to indicate destroyed restriction sites.

membrane spanning domains and should be present in the membrane based on the PhoAþ phenotype of an ORF3–PhoA fusion. However, the charge distribution is not agreement with the requirements for holins (Young, 1992). Therefore, it is unlikely that a typical holin is encoded in the region surrounding atlA . Inverted repeat sequences that could form stem–loop structures were identified upstream of atlA and downstream of orf2 (Fig. 3). Two possible ribosome-binding sites (RBSs) for AtlA were found in the region between the inverted repeats in the upstream stem–loop. This finding is intriguing as the stem–loop structure encompassing the two RBSs of the lambda holin gpS is used to regulate lysis (Young, 1992). Neither of the stem–loop structures contained a gonococcal transformation uptake sequence (GCU) (Goodman and Scocca, 1988), which is often found in gonococcal terminators. However, a sequence with 9 out of 10 matches to the GCU is found within the coding region. No sequences similar to the sigma-70

type promoter consensus sequence were detected in the region upstream of atlA , suggesting that either atlA uses an alternate sigma factor or that its promoter is upstream of orf1.

Phenotypic characterization of an atlA mutant To assess the function of the putative autolysin, we constructed insertion/deletion mutants. An erythromycin resistance marker, ermC, was used to replace the 38 end of atlA in plasmid clone pJD1103. Insertions were obtained in both orientations, and the resulting constructs were used to transform N. gonorrhoeae strain MS11 A. Southern analysis of chromosomal DNA from transformants JD1510 and JD1511 showed these strains contained the expected mutations with the truncated atlA8 and the ermC marker having replaced the wild-type gene in the forward or reverse direction respectively (Fig. 2). In JD1510 the inserted ermC gene is transcribed in the

Fig. 3. DNA and deduced amino acid sequence of the atlA region. The sequence is numbered beginning with the stop codon for ORF1. Putative RBSs for atlA and orf2 are underlined. Regions of dyad symmetry are indicated by arrows. The start codon for atlA was presumed to be the first start codon within the atlA open reading frame adjacent to a predicted RBS. A sequence with nine out of ten matches to the GCU is underlined in bold. Q 1997 Blackwell Science Ltd, Molecular Microbiology, 25, 893–901

896 J. P. Dillard and H. S. Seifert

Fig. 4. PG turnover. PG turnover was measured as loss of labelled PG from the macromolecular fraction of gonococci grown in liquid culture after pulse labelling with 6-[ 3H]-glucosamine. Values are given as a percentage of radioactive counts relative to that at the beginning of the chase period and are the average of three experiments. Error bars represent the standard deviations.

same direction as orf2, and transcription is likely to proceed through orf 2. Thus, the mutation in JD1510 is probably not polar on orf2 transcription. However, the translation start codon for orf2 is close to the translation stop for atlA , suggesting that the two genes may be translationally coupled. Thus, we cannot rule out the possibility that the atlA mutations have polar effects on orf2 expression and may be partly responsible for phenotype of the mutants. JD1510 and JD1511 were used in all the analyses described below (except electron microscopy) and gave similar results; therefore, only the results for JD1510 are shown. One role of PG hydrolysing enzymes is in the degradation and turnover of the cell wall during growth. The rates of PG turnover were compared between the mutant and wild-type strains to determine if the mutation had an effect on PG turnover (Fig. 4). The PG of the two strains was pulse-labelled with 6-[ 3H]-glucosamine. Mutant and wildtype strains labelled equally well. The rate of PG turnover was defined as the rate at which labelled material was lost from the cell wall fraction. The rate of turnover was the same for the mutant and wild-type strains during log phase. However, upon entry into stationary phase, PG turnover in the mutant stopped for a period of 8 h, whereas the wild-type strain merely slowed its PG turnover rate. The mutant and wild type showed the same rate of turnover in late stationary phase, but the mutant constantly maintained more PG throughout stationary phase. Consistent with the PG turnover results, the atlA mutation had no obvious effect on exponential growth – the doubling time in liquid culture was equivalent to the wildtype strain. The doubling times of the mutant at pH 6.2,

7.2 or 8.2 were the same as that of the wild-type strain. The colony morphologies of both piliated and non-piliated variants appeared normal. The mutant had similar numbers of diplococci and single cocci compared with the wild-type strain when examined by light microscopy (data not shown). Thus, AtlA is not essential for growth or septation and is not involved in determining cell shape. In fact, there were no observable differences between the mutant and wild-type strain during growth. Previous studies characterizing autolysis in N. gonorrhoeae have measured autolysis by following the decrease of optical density (OD) after the cells were transferred to non-growth conditions (Hebeler and Young, 1975; Elmros et al., 1976a; Wegener et al., 1977). We measured changes in OD after transferring cultures into Tris-buffered solution to determine whether the atlA mutation affected this type of lysis. The amidase and endopeptidase activities had been shown to function at pH 8 but were inhibited at pH 6 (Hebeler and Young, 1975; Chapman and Perkins, 1983). We therefore performed the assay at pH 6 and pH 8 to determine whether AtlA-mediated activity could be differentiated from the activities of the amidase and endopeptidase. At the amidase/endopeptidase permissive condition (pH 8) the mutant and wild-type strains showed identical high rates of autolysis as measured by OD decrease (Fig. 5). However, at the amidase/endopeptidase nonpermissive condition (pH 6), the AtlA mutant showed an initial drop in OD, which presumably was owing to the shift from media to buffer, but showed only a slight decrease in turbidity thereafter. In contrast, the parental strain showed

Fig. 5. Autolysis in buffer. Autolysis was measured as the change in optical density at 540 nm after transfer to 50 mM Tris-HCl buffer at pH 6 (open symbols) or pH 8 (closed symbols). Values are represented as a percentage of starting culture density and are the average of three experiments. Error bars represent the standard deviations. Q 1997 Blackwell Science Ltd, Molecular Microbiology, 25, 893–901

Autolysin of N. gonorrhoeae 897

Fig. 6. Autolysis in liquid culture. Growth and lysis in liquid culture were measured by following both OD and viability. Gonococcal cultures in early stationary phase (OD540 of 1.5) were diluted to an OD540 of 0.2 in GCBL, aliquoted into 3 ml cultures and grown with shaking at 378C. At the indicated times, 1 ml was measured for OD (A), and 20 ml was serially diluted and plated on GCB agar plates for CFU determinations (B). One representative experiment is shown. In 11 experiments of this type, the dramatic death occurred nine times. The limit of detection for viable counts was 500 ml¹1, and all undetectable values were represented as 500.

a constant drop in turbidity throughout the course of the assay. Therefore, the atlA mutant is deficient in an autolytic activity that is distinct from the amidase and endopeptidase activities. Gonococci are notoriously fragile and do not survive under several common laboratory conditions, including growth in the stationary phase (Morse and Bartenstein, 1974; Elmros et al., 1976a). As gonococci have been shown to express at least three autolytic activities (Hebeler and Young, 1976; Sinha and Rosenthal, 1980; Chapman and Perkins, 1983), the rapid decrease in viability in stationary phase is thought to be due to autolysis. The timing of the fall can be affected by the amount of carbon source supplied, suggesting that the cells begin to lyse when nutrients are exhausted (Morse and Bartenstein, 1974). Therefore, we tested the effect of the atlA mutation on stationary phase survival. The OD of the wild-type and atlA strain were followed for 40 h after stationary phase was reached (Fig. 6 A). During stationary phase, the wild-type strain decreased in OD by about one-quarter its highest value, dropping from 1.6 to 1.2 OD540. The mutant maintained a consistently higher density throughout stationary phase. Both strains showed a small reproducible increase in OD at about 24 h into stationary phase. A much more dramatic effect of the atlA mutation was found by monitoring viable CFUs over extended time in stationary phase (Fig. 6B). Again, the mutant strain maintained its viability much better than the wild-type strain in stationary phase, showing threefold more viable counts than the wild-type strain at 21 h. After a reproducible increase in CFUs for both the strains, the wild-type strain showed a dramatic reduction in viability, decreasing more than 5 000-fold in less than 2 h. The mutant strain also died after the stationary phase growth, but took more than 20 h to reach a 5 000-fold reduction of viability. Q 1997 Blackwell Science Ltd, Molecular Microbiology, 25, 893–901

Transmission electron microscopy was used to confirm that the changes in OD truly represented cell lysis. In exponential phase growth (2.5 h) the mutant and wild-type cells appeared identical. The cells stained darkly and had welldefined edges (data not shown). Differences in the degree of lysis between the mutant and wild-type strains could be seen in electron micrographs from stationary phase cultures (42 h). Unusual staining and irregularly shaped forms, presumably lysed cells, could be seen in the stationary phase cultures and predominated in the wildtype culture. Most of the lysed cells were lightly stained but had dark-staining spots. These cells appeared as smudges with no defined edges. At 42 h, no intact cells could be found in the wild-type culture, whereas the mutant culture contained some lysed cells but still contained many intact cells as well. Taken together, these results strongly suggest that AtlA plays a major role in survival in stationary phase and that the mutant is more resistant to both cell lysis and death in stationary phase. Discussion The data presented here indicate that atlA encodes a PG hydrolase. The sequence similarity suggests that AtlA is a PG transglycosylase, and thus is likely to have an activity that has been detected previously in gonococci (Sinha and Rosenthal, 1980). A mutant containing a truncated version of atlA is inhibited in lysis during stationary phase and when transferred into buffer. During growth in liquid culture, the atlA mutant was identical to the wild-type strain in exponential phase growth, but was less lytic in stationary phase and maintained more viable counts. In addition, the mutant did not suffer the catastrophic death seen in the wild-type culture. The rate of PG turnover was reduced in the mutant relative to that of the wild

898 J. P. Dillard and H. S. Seifert type during early stationary phase. As the atlA mutant is reduced in lysis in buffer, lysis in culture, death in culture and turnover of PG, AtlA is probably a PG hydrolase. The phenotypes we observed in the AtlA mutant occurred during stationary phase, with no function of AtlA apparent during normal growth. Thus, AtlA may only act during stationary phase or under other conditions in which autolysis could be advantageous to the bacterial population. Alternatively, another enzyme may substitute for AtlA in the mutant during growth, as was seen for the PG hydrolysing lytic transglycosylases involved in cell wall growth in E. coli (Templin et al., 1992; Ehlert et al., 1995). E. coli produces at least three lytic transglycosylases (Holtje and Tuomanen, 1991), and mutants lacking either of two of these enzymes show no defect in growth (Templin et al., 1992; Ehlert et al., 1995). One of these enzymes (Slt70) was shown to associate with PBP3 and PBP7/8, and is thought to act in removing old PG strands as the new PG strands are inserted, thus facilitating cell wall expansion (Romeis and Holtje, 1994). If AtlA is involved in cell wall growth and a second enzyme complements its function in the mutant during growth, this second enzyme must not function in the stationary phase culture or at acidic pH. One interesting aspect of autolysis in N. gonorrhoeae is that the cells do not completely disintegrate as is seen in lambda-mediated lysis of E. coli (Young, 1992). The ODs of the cultures show only moderate decreases, and lysed cells still have some shape and size similar to intact cells. One explanation for this stability is that Mg2þ, which is present in the growth medium, stabilizes the outer membrane lending strength to a broken cell wall (Wegener et al., 1977). Another possibility is that the normal function of AtlA is not to degrade whole strands of PG but rather to produce localized breaks in the PG. It has recently been recognized that a family of PG hydrolases, made up of both PG transglycosylases and true lysozymes, is involved in secretion of macromolecules (Mushegian et al., 1996), presumably by introducing small holes in the cell wall. The list of macromolecules using PG hydrolysis for export is extensive, but includes flagella, type III secretion apparatus, and conjugative and Ti plasmid DNA (Dijkstra and Keck, 1996; Mushegian et al., 1996). If AtlA acts to create small holes in the cell wall, then its effects on autolysis may be more indirect, i.e. mutant cells lacking these pores may have more mechanical stability than wild-type cells in stationary phase culture. Autolysis has been described as part of the bacterium’s ‘catastrophe kit’, a collection of functions that bacterial populations use to avoid death under severely adverse conditions (Koch, 1996). Thus, autolysis may provide some advantages to the infecting bacterial population (if not to the lysing bacterium). Under starvation conditions, the lysing bacteria can provide nutrients to the unlysed

portion of the population, perhaps allowing them to live long enough to escape calamity. Autolysis may be necessary for donating DNA for natural transformation. By undergoing transformation, a recombinant may be formed that is able to survive under the adverse conditions or avoid immune surveillance. As AtlA is capable of destroying the cell, its regulation is crucial. We do not know how AtlA expression is regulated, but examination of the growth curve suggests that AtlA activity is regulated during stationary phase (Fig. 6). Two distinct phases characterized by different degrees of lysis are evident in stationary phase. In early stationary phase, there was considerable cells lysis and cell death in the wild-type culture, but the mutant was substantially protected from both lysis and death. After an extended time in stationary phase, both the mutant and wild-type cultures showed reproducible increases in OD and CFU. Surprisingly, these increases must reflect one or more rounds of cell division that occur more than 16 h after the entry into stationary phase. This could reflect the rise of a subpopulation that has a growth advantage under starvation conditions, as has been observed in E. coli (Zambrano and Kolter, 1996), but this type of phenomenon has not been described in N. gonorrhoeae. After the replication event, the wild-type strain died quickly, whereas the mutant died very slowly. We do not know how growth is initiated after such an extended time in the stationary phase. However, we have observed that the timing of the growth and death can be affected by changing the amount or type of carbon source supplied. It is also notable that although the growth and dramatic death generally occurs as described, it occasionally does not occur in a culture. This suggests the requirement for an initiating event, and that that event is controlled in a stochastic manner. Although we have no evidence that it is involved in regulation, we have identified a putative stem–loop structure located just 58 of the coding region that could be involved in AtlA expression. As it encompasses one of two potential RBSs, it could affect translational initiation or, alternatively, it might affect transcription. Because of the sequence similarity between AtlA and the phage endolysins, it is tempting to speculate that atlA is part of an integrated bacteriophage. However, the sequence similarity of AtlA to the bacteriophage endolysins are the only data favouring this conclusion. The ORFs in the surrounding region do not have to have the structural characteristics of a holin and show no homology to other bacteriophage proteins. The presence of the cold shock protein homologue also argues against the idea that atlA is part of a phage, as cold shock proteins are commonly encoded in bacterial genomes (Jones and Inouye, 1994) but have not been reported in bacteriophage genomes. Moreover, multiple attempts to identify phages in N. gonorrhoeae, have been unsuccessful (Stone et al., Q 1997 Blackwell Science Ltd, Molecular Microbiology, 25, 893–901

Autolysin of N. gonorrhoeae 899 1956; Phelps, 1967; Steinberg et al., 1976). Therefore, the evidence suggests that atlA is a bacterial homologue of the phage endolysins that functions to promote cell lysis during the stationary phase of growth.

Experimental procedures

Bacterial strains and culture conditions Derivatives of N. gonorrhoeae strain MS11A were used in these studies (Segal et al., 1985). All experiments were performed with non-piliated variants except where indicated. Strains JD1510 and JD1511 have the ermC marker replacing the 38 end of atlA . Gonococci were grown with shaking in GCB liquid medium (Difco) plus Kellogg supplements (GCBL) (Kellogg et al., 1963) and 0.042% NaHCO3 (Morse and Bartenstein, 1974) or on GCB agar plates in 5% CO2 at 378C. GCBL contains 0.4% glucose. E. coli strain DH5a (BRL) was used routinely. E. coli strains were grown in Luria broth or on Luria agar plates (Sambrook et al., 1989). Kanamycin at 40 mg ml¹1 was used for E. coli containing pHSS6 (Seifert et al ., 1986) derivatives. Erythromycin was used at 250 mg ml¹1 for E. coli and 10 mg ml¹1 for N. gonorrhoeae to select for expression of ermC.

Table 1. Plasmids. Plasmid

Properties

Source or reference

pCBB 1

Hpa II-Sau 3AI fragment from MS11 A in pHSS7 with mTnCmPhoA in orf3 cloning vector, KmR ermC in pHSS6 cloning vector, ApR

Boyle-Vavra and Seifert (1994)

pHSS6 pHSS25 pUC19 pJD1101 pJD1102 pJD1103 pJD1104 pJD1105

pJD1108

pJD1133

pJD1134

pCBB1 D 5.5 kb Mfe I fragment pCBB1 D 6 kb MfeI-EcoRI fragment 0.5 kb Eco RI-Mfe I fragment from pCBB1 in pHSS6 0.4 kb Eco RI* fragment from pCBB1 in pHSS6 2.2 kb Sau 3AI fragment from MS11 A in pHSS7 overlapping pCBB1 at orf4 2.4 kb Sau 3AI fragment from MS11 A in pHSS7 overlapping pCBB1 at orf4 1.2 kb Sma I-Eco RI ermC fragment from pHSS25 replacing 134 bp Dra I fragment in pJD1103 as pJD1133, ermC orientation reversed

Seifert et al. (1986) Wainwright et al. (1994) Yannish-Perron et al. (1985) This work This work This work This work This work

This work

This work

This work

N. gonorrhoeae transformation Gonococcal transformation was performed by incubating 5 × 107 piliated gonococci in 200 ml of GCB containing 5 mM MgSO4 with excess donor DNA for 5 min at 378C (Seifert et al., 1990). Cells were diluted 1:10 into GCBL and allowed to grow 3–5 h before plating.

Plasmids The plasmids used in this study are listed in Table 1. The plasmid clone pCBB1 contains a 1.6 kb fragment from MS11 A with a mTnCmPhoA insertion in orf3. It was identified from the CBB library (Seifert and Wilson, 1992) as a clone giving a PhoAþ phenotype in E. coli and N. gonorrhoeae (S. Boyle-Vavra and H. S. Seifert, unpublished results). Plasmid clones pJD1105 and pJD1108 were identified from library CBB by Southern blotting using pCBB1 as the probe. Plasmids pJD1133 and pJD1134 are derivatives of pJD1103 with ermC replacing the 38 134 bp of atlA coding sequence. A 1.2 kb EcoR I– Sma I fragment containing the ermC gene was excised from pHSS25 (Wainwright et al., 1994), bluntended using Klenow and ligated into the Dra I sites of pJD1103. Plasmid clones pJD1133 and pJD1134 contain the ermC gene in the same and opposite orientations, respectively, relative to the direction of atlA transcription.

probes made by random priming using [a-32P]-dCTP (Amersham). Searches of the NCBI databases were performed using the Blastp and Blastx programs (Altschul et al., 1990). Pairwise alignments were performed using the PALIGN program, and multiple sequence alignments were performed with the CLUSTAL program of the PCGENE suite (Inteligenetics). Fragments for sequencing upstream of atlA , which we were unable to clone, were isolated from the N. gonorrhoeae MS11 A chromosome by PCR amplification. Chromosomal digests were run on a TAE gel, and fragments of the predicted size, based on Southern analyses, were excised, purified using Geneclean (Bio101) and ligated to pHSS6. The desired fragments were obtained by PCR amplification using primers for the vector and atlA . The DNA sequence data have been submitted to the GenBank database under accession number AF010308.

Autolysis in buffer Gonococcal cultures (3 ml) in early stationary phase were centrifuged at 3000 × g for 8 min and the cells were resuspended at an OD540 of 0.2 in 50 mM TrisHCl buffer at pH 6 or pH 8. Cells were kept at 258C and measured for OD540 at the indicated times.

DNA techniques DNA sequencing was performed using Sequenase and [a-35S]-dATP (Amersham). Southern blotting was performed by standard techniques (Sambrook et al., 1989). DNA was transferred using a pressure blotter and UV cross-linked to nylon membranes (Stratagene). Blots were hybridized with Q 1997 Blackwell Science Ltd, Molecular Microbiology, 25, 893–901

PG turnover To label the PG, gonococci were grown for 2 h in GCBL containing 0.4% pyruvate and 6-[ 3H]-glucosamine (New England Nuclear) at 2 mCi ml¹1 (Wegener et al., 1977). Cells were washed with GCBL to remove unincorporated label. Cells

900 J. P. Dillard and H. S. Seifert were resuspended in GCBL (containing glucose at 0.4%) at an OD540 of 0.2 and allowed to grow for the indicated times. The macromolecular PG was isolated by a modification of the procedure of Rosenthal (Rosenthal and Dziarski, 1994). Cultures (3 ml) were centrifuged at 3000 × g for 6 min at 08C, resuspended in 0.5 ml of 50 mM sodium acetate buffer at pH 5, and lysed by the addition of an equal volume of 8% sodium dodecyl sulphate. The lysates were boiled for 30 min, and the macromolecular PG was pelleted using centrifugation for 30 min at 18000 × g. The PG was resuspended in 0.5 ml of water and the amount of the original PG remaining was determined by liquid scintillation counting.

Electron microscopy Gonococcal cultures were centrifuged (2600 × g, 5 min) onto poly L-lysine-treated, formvar-coated copper grids (Ladd). The cells were fixed in 1% glutaraldehyde in 0.1 m sodium cacodylate buffer pH 7.4 for 2 min, washed twice with H2O and negatively stained with 1% phosphotungstic acid pH 6.0 for 1 min (McGee et al., 1976). Grids were examined at 60 kV in a Jeol JCM-10 CX II electron microscope.

Acknowledgements We thank Nick Cianciotto and Jim Duncan for a critical reading of the manuscript. This work was supported by Grant AI33493 to H.S.S. from the National Institutes of Health. J.P.D. was supported by NRSA Fellowship AI09347.

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