Sialylation of group B streptococcal capsular polysaccharide is mediated by cpsK and is required for optimal capsule polymerization and expression.

Several bacterial pathogens have evolved the means to escape immune detection by mimicking host cell surface carbohydrates that are crucial for self/non-self recognition. Sialic acid, a terminal residue on these carbohydrates, inhibits activation of the alternate pathway of complement by recruiting the immune modulating molecule factors H, I, and iC3b. Sialylation of capsular polysaccharide (CPS) is important for virulence of group B streptococci (GBS), a significant human pathogen. We previously reported that cpsK, a gene within the cps locus of type III GBS, could complement a sialyltransferase deficient lst mutant of Haemophilus ducreyi, implicating its role in sialylation of the GBS capsule. To explore the function of cpsK in GBS capsule production, we created a mutant in cpsK. Immunoblot analysis and enzyme-linked immunosorbent assay using anti-type III CPS antisera demonstrated that the mutant CPS did not contain sialic acid. This was confirmed by high-performance liquid chromatography after mild acid hydrolysis of the CPS. Although increased CPS chain length was seen for this strain, CPS production was <20% of the parental isolate. An episomal cpsK copy restored synthesis of sialo-CPS to wild-type levels. These data support our hypothesis that cpsK encodes the GBS CPS sialyltransferase and provide further evidence that lack of CPS oligosaccharide sialylation reduces the amount of CPS expressed on the cell surface. These observations also imply that one or more of the components involved in synthesis or transport of oligosaccharide repeating units requires a sialo-oligosaccharide for complete activity.

NeuD plays a role in the synthesis of sialic acid in Escherichia coli K1.

The polysialic acid capsule of Escherichia coli K1 is an essential virulence determinant. The kps gene cluster, which encodes the proteins necessary for polymer synthesis and transport, is divided into three functional regions. In this report, we present evidence that the neuD gene from region 2 is involved in sialic acid synthesis. A non-polar chromosomal deletion in neuD was constructed. The defect was complemented by neuD in trans or by the addition of exogenous sialic acid. A NeuD homologue, Neu(III)D, from serotype III Streptococcus agalactiae (GBS) also restored capsule expression to the neuD deletion strain. These data confirm the role of neuD in E. coli sialic acid capsule synthesis and demonstrate that the neu(III)D homologue from GBS shares a similar enzymatic function.

The serotype of type Ia and III group B streptococci is determined by the polymerase gene within the polycistronic capsule operon.

Streptococcus agalactiae is a primary cause of neonatal morbidity and mortality. Essential to the virulence of this pathogen is the production of a type-specific capsular polysaccharide (CPS) that enables the bacteria to evade host immune defenses. The identification, cloning, sequencing, and functional characterization of seven genes involved in type III capsule production have been previously reported. Here, we describe the cloning and sequencing of nine additional adjacent genes, cps(III)FGHIJKL, neu(III)B, and neu(III)C. Sequence comparisons suggested that these genes are involved in sialic acid synthesis, pentasaccharide repeating unit formation, and oligosaccharide transport and polymerization. The type III CPS (cpsIII) locus was comprised of 16 genes within 15.5 kb of contiguous chromosomal DNA. Primer extension analysis and investigation of mRNA from mutants with polar insertions in their cpsIII loci supported the hypothesis that the operon is transcribed as a single polycistronic message. The translated cpsIII sequences were compared to those of the S. agalactiae cpsIa locus, and the primary difference between the operons was found to reside in cps(III)H, the putative CPS polymerase gene. Expression of cps(III)H in a type Ia strain resulted in suppression of CPS Ia synthesis and in production of a CPS which reacted with type III-specific polyclonal antibody. Likewise, expression of the putative type Ia polymerase gene in a type III strain reduced synthesis of type III CPS with production of a type Ia immunoreactive capsule. Based on the similar structures of the oligosaccharide repeating units of the type Ia and III capsules, our observations demonstrated that cps(Ia)H and cps(III)H encoded the type Ia and III CPS polymerases, respectively. Additionally, these findings suggested that a single gene can confer serotype specificity in organisms that produce complex polysaccharides.

Blue/white screening of recombinant plasmids in Gram-positive bacteria by interruption of alkaline phosphatase gene (phoZ) expression.

The process of screening bacterial transformants for recombinant plasmids is made more rapid and simple by the use of vectors with visually detectable reporter genes. In such systems, an alteration in colony phenotype occurs when a vector-borne indicator gene is interrupted with exogenous DNA. Although the lacZ system has been used extensively for this purpose in E. coli, analogous systems for use in Gram-positive bacteria remain uncommon. We have developed a Gram-positive cloning vector that utilizes the interruption of an alkaline phosphatase gene, phoZ, to identify recombinant plasmids. To facilitate introduction of foreign DNA, a multiple cloning site (MCS) was inserted distal to the region coding for the putative signal peptide of phoZ. Alkaline phosphatase expressed from the derivative phoZ gene (phoZMCS) retained activity similar to that of the native protein. The phoZMCS was transferred to pJS3, a well-characterized, high-copy number, and broad-host-range plasmid, to produce pDC123. In pDC123, phoZMCS was transcriptionally linked to the chloramphenicol acetyl transferase (cat) gene under the control of the constitutively expressed tetM and cat promoters that drive cat expression in pJS3. S. agalactiae (Group B streptococci, GBS), E. faecalis, S. pyogenes, S. gordonii, and E. coli containing pDC123 displayed a blue colonial phenotype on agar containing 5-bromo-4-chloro-3-indolyl phosphate (X-p), which was readily distinguished from that of colonies containing the parent plasmid pJS3. Introduction of foreign DNA into the MCS of phoZMCS produced a white colonial phenotype in E. coli and GBS on agar containing X-p and allowed discrimination between transformants containing recombinant plasmids versus those maintaining self-annealed or uncut vector. We have used pDC123 to subclone the cpsE gene from the plasmid pCER111, which carries a 9.0-kb fragment of the GBS capsular polysaccharide synthesis locus. The plasmid pDC123 containing cpsE was isolated by direct electroporation into GBS strain A909 with selection of transformants containing recombinant plasmids achieved by 'blue/white' screening, without the use of an intermediate host. This new cloning vector should improve the efficiency of performing recombinant DNA experiments in Gram-positive bacteria.

Trimethoprim resistance in Haemophilus influenzae is due to altered dihydrofolate reductase(s).

We characterized a highly purified preparation of the chromosomally encoded dihydrofolate reductase (DHFR) from a trimethoprim-susceptible (Tmp8; strain MAP) and two trimethoprim-resistant (TmpR) strains (MAP/47 and MAP/42) of Haemophilus influenzae. The enzymes were purified between 650- and 3000-fold by gel-filtration and dye-ligand chromatography. The apparent molecular mass of the three proteins was 18400 Da by PAGE under denaturing and nondenaturing conditions. Total enzyme activity was greater in all fractions from the TmpR strains compared with the Tmp8 isolate. The three enzymes had a similar Km for dihydrofolate (7, 9 and 5 microM) and NADPH (2, 5 and 6 microM). However, the Tmp IC50 (the concentration necessary for 50% inhibition of DHFR activity) for the Tmp8 strain MAP was 0.001 microM, whereas DHFR from the TmpR strains MAP/47 and MAP/42 had values of 0.1 microM and 0.3 microM respectively. The methotrexate IC50 of the MAP/42 DHFR was 0.06 microM in comparison with the enzyme from MAP (0.008 microM) and MAP/47 (0.007 microM). Isoelectric focusing indicated that the DHFR from MAP/42 had a different isoelectric point (pI 7.6) compared with the enzymes from MAP and MAP/47 (pI 7.3). Peptide mapping after digestion with trypsin revealed one major peptide fragment (7.9 kDa) in the DHFR of MAP and MAP/47 and three major tryptic fragments (7.9, 9.6 and 12.5 kDa) in DHFR from MAP/42. We conclude that trimethoprim resistance in H. influenzae results from overproduction of structurally altered DHFR(s).

Cefuroxime treatment failure of nontypable Haemophilus influenzae meningitis associated with alteration of penicillin-binding proteins.

A 10-year-old boy presented with nuchal rigidity and cerebrospinal fluid (CSF) leukocytosis initially and again on day 6 of intravenous cefuroxime therapy (200 mg/kg/day). Both CSF specimens yielded nontypable beta-lactamase-negative Haemophilus influenzae that were susceptible by disk tests but relatively resistant to cefuroxime (MIC, 8- to 16-fold greater than that of control isolates). To define the mechanism of resistance, the cefuroxime resistance marker was transformed to a susceptible H. influenzae recipient; inactivation and permeability of beta-lactam substrate were tested and the penicillin-binding protein (PBP) profiles were examined. Inactivation of beta-lactam substrate was not detected and reduced permeability was not found. However, reduced beta-lactam binding to PBPs 4 and 5 was observed; 18- to 27-fold more penicillin and 2.5-to 4-fold more cefuroxime was required to saturate or block 50% of the binding sites of these PBPs, respectively. Thus, reduced affinity of PBPs 4 and 5 for beta-lactam substrate appears to be the mechanism of cefuroxime resistance in this strain. The reduced affinity of these targets appears to have contributed to the bacteriologic and clinical failure in this patient.

Problems with current recommendations for susceptibility testing of Haemophilus influenzae.

We compared results of MIC and disk susceptibility tests on Haemophilus test medium (HTM) and those on comparative media. Ampicillin MICs were determined with seven ampicillin-resistant, non-beta-lactamase-producing (AmprNBLP) isolates by using HTM and supplemented brain heart infusion (sBHI) agar. Ampicillin and amoxicillin-clavulanate disk tests with 16 AmprNBLP strains, 18 ampicillin-susceptible (Amps) isolates, and 17 ampicillin-resistant, beta-lactamase-producing (AmprBLP) strains were performed by using five media: laboratory-prepared HTM (PHTM), commercial HTM (CHTM), sBHI, enriched chocolate agar, and Mueller-Hinton chocolate agar. We observed that five of seven and three of seven AmprNBLP strains were misclassified as susceptible with PHTM (MIC, less than 2 micrograms/ml) with inocula of 10(3) and 10(5) CFU, respectively, but were resistant with sBHI (MIC, greater than or equal to 2 micrograms/ml). Whereas Mueller-Hinton chocolate agar and enriched chocolate agar plates supported the growth of all 51 strains by the disk tests, 37% (19 of 51) and 8% (4 of 51) of strains did not grow on PHTM and CHTM, respectively. Lack of growth on PHTM was observed for all three phenotypes; 7 of 18 Amps, 4 of 17 AmprBLP, and 8 of 16 AmprNBLP strains did not grow. The four strains that did not grow on CHTM were all AmprNBLP isolates. Zone sizes were significantly larger on PHTM than on the other media. Of the strains that were evaluable by the new National Committee for Clinical Laboratory Standards guidelines with either PHTM or CHTM, all Amps strains were classified as susceptible. Among the AmprBLP strains, CHTM correctly identified all as resistant, whereas PHTM detected two isolates to be intermediate. Among the AmprNBLP strains, CHTM and PHTM misclassified four (33%) and five (62%) isolates, respectively, as susceptible; an additional isolate was identified as intermediate on both media. We conclude that there is strain-dependent growth on HTM, that adoption of this medium for routine Haemophilus susceptibility testing is problematic due to this growth variability, and that detection of AmprNBLP isolates would be unreliable.

Penicillin-binding proteins and ampicillin resistance in Haemophilus influenzae.

Ampicillin-resistant, non-beta-lactamase-producing isolates of Haemophilus influenzae contain a variety of penicillin-binding protein (PBP) patterns that differ from the single pattern of eight PBPs characteristic of susceptible strains. During genetic transformation of resistance, only some of the anomalies in PBP pattern were transformed, specifically those relating to the penicillin-binding capacities of PBPs 4 (Mr of 62,000) and 5 (Mr of 59,000) and, in some transformations, PBP 3 (Mr of 71,000). Comparison of the binding of penicillin by PBPs 4 and 5 of three resistant transformants (derived with DNA from different donors) revealed a decrease in the rate of PBP acylation and no appreciable change in the rate of deacylation as compared to the susceptible recipient. Thus, rapid turnover of these PBPs does not play a role. Retransformation studies confirm that altered PBPs 3, 4, and 5 are associated with resistance and suggest that these PBPs are major targets for the beta-lactam antibiotics in H. influenzae.

In vitro activities and targets of three cephem antibiotics against Haemophilus influenzae.

The antimicrobial activities of cefixime, cefpodoxime, and ceftibuten were determined with 18 ampicillin-susceptible (Amps), 13 ampicillin-resistant beta-lactamase-producing (AmprBLP), and 7 ampicillin-resistant non-beta-lactamase-producing (AmprNBLP) strains of Haemophilus influenzae. An effect of inoculum density on apparent MIC, the bactericidal activity of these agents, and the targets of the three cephems were determined. The MICs of cefixime, cefpodoxime, and ceftibuten for 90% of the Amps and AmprBLP isolates were 0.04, 0.08, and 0.08 microgram/ml, respectively. In contrast, the MICs for 90% of the AmprNBLP strains were 0.96, 1.92, and 7.68 micrograms/ml. No significant inoculum effect was observed for any group of strains comparing inocula of 10(3) and 10(5) CFU, whereas only the AmprNBLP isolates showed a marked effect at an inoculum of 10(6) CFU. Although bactericidal levels were achieved for the Amps and AmprBLP strains, tolerance to cefixime and ceftibuten was observed. The bactericidal activity for the AmprNBLP strains was limited, with cefixime showing the highest activity of the three cephems. Penicillin-binding proteins 2, 4, and 5 revealed high affinity, with 50% inhibitory concentration levels below the MIC for all three cephems, suggesting that these are important targets of these agents in H. influenzae. We conclude that the cephems are highly active in vitro against Amps and AmprBLP strains of H. influenzae, but less so against AmprNBLP isolates.

PBP profiles of Haemophilus influenzae, H. aegyptius, and the H. influenzae biogroup aegyptius associated with Brazilian Purpuric Fever.

We questioned if PBP analysis could differentiate strains of Haemophilus influenzae, H. aegyptius, and H. influenzae biogroup aegyptius associated with Brazilian Purpuric Fever. A relatively homogeneous PBP pattern was observed for all strains. The amount of penicillin bound to PBP 5 appeared to separate H. influenzae and H. aegyptius isolates, whereas PBP 5 of those strains associated with Brazilian Purpuric Fever bound an intermediate amount. We conclude that based on PBP profiles, the strains tested appear to be difficult to separate taxonomically and may represent a common species.

Statistical comparison of ligand-binding kinetics.

We describe the application of generalized linear model methodology to the problem of testing differences among ligand-receptor interactions, and show that the method is analogous to weighted least squares regression methodology and F tests familiar to many investigators. The method accommodates incomplete block designs so that one can obtain kinetic parameter estimates directly comparable among samples analysed on incompletely overlapping sets of experimental runs. We demonstrate the method with data that compare saturation kinetics for a single penicillin-binding protein in isogenic ampicillin susceptible and resistant bacteria.

Genetic diversity of penicillin G-resistant Neisseria meningitidis from Spain.

Genotypic and phenotypic diversity among 16 penicillin G-resistant (Penr) isolates of Neisseria meningitidis recovered from human blood or cerebrospinal fluid in Spain was compared with that among 12 penicillin-susceptible (Pens) isolates by the use of multilocus enzyme electrophoresis, serotyping, auxotroph testing in chemically defined media, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of penicillin-binding proteins (PBPs). Thirteen distinctive multilocus enzyme genotypes (electrophoretic types [ETs] ) were identified among the 28 isolates. There was slightly less genetic diversity among the eight ETs of Penr isolates (H = 0.385) than among the eight ETs of Pens isolates (H = 0.431). Cluster analysis demonstrated two distinctive complexes of ETs and one ET that was not closely related to either complex. The possibility of a singular clonal origin of penicillin G-resistant isolates was excluded by the observations that resistance occurred in isolates of each of the two distantly related complexes of ETs, that three of the four ETs represented by multiple isolates included both susceptible and resistant strains, and that serotypes and growth requirements were not associated with the resistance phenotype. The 28 isolates showed a relatively homogeneous pattern of four PBPs, with apparently reduced penicillin G binding by PBP 3 of the Penr isolates.

Relative penicillin G resistance in Neisseria meningitidis and reduced affinity of penicillin-binding protein 3.

We examined clinical isolates of Neisseria meningitidis relatively resistant to penicillin G (mean MIC, 0.3 micrograms/ml; range, 0.1 to 0.7 micrograms/ml), which were isolated from blood and cerebrospinal fluid for resistance mechanisms, by using susceptible isolates (mean MIC, less than or equal to 0.06 micrograms/ml) for comparison. The resistant strains did not produce detectable beta-lactamase activity, otherwise modify penicillin G, or bind less total penicillin. Penicillin-binding protein (PBP) 3 of the six resistant isolates tested uniformly bound less penicillin G in comparison to the same PBP of four susceptible isolates. Reflecting the reduced binding affinity of PBP 3 of the two resistant strains tested, the amount of 3H-labeled penicillin G required for half-maximal binding was increased in comparison with that of PBP 3 of the two susceptible isolates. We conclude that the mechanism of resistance in these meningococci relatively resistant to penicillin G was decreased affinity of PBP 3.

Genetic and phenotypic diversity among ampicillin-resistant, non-beta-lactamase-producing, nontypeable Haemophilus influenzae isolates.

Levels of genotypic and phenotypic diversity among 23 ampicillin-resistant, non-beta-lactamase-producing (Ampr NBLP) isolates of serologically nontypeable Haemophilus influenzae recovered from the respiratory tract were determined by multilocus enzyme electrophoresis, auxotroph testing in chemically defined media, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of penicillin-binding proteins (PBPs). Twenty distinctive multilocus enzyme genotypes were identified, among which the average level of genetic diversity per locus was equivalent to that in the species as a whole. Hence, a single, recent origin for Ampr NBLP strains is excluded. Of the growth factors tested, a requirement for methionine was significantly associated with the Ampr NBLP phenotype. In contrast to the relative homogeneity of the PBP profiles of the ampicillin-susceptible strains tested (8 PBPs detected), the PBP profiles of the Ampr NBLP strains exhibited marked heterogeneity (5 to 10 PBPs detected). Care should be taken in interpreting changes in PBP profiles and in associating these profiles with resistance for species such as H. influenzae that demonstrate variability.

Detection of relatively penicillin G-resistant Neisseria meningitidis by disk susceptibility testing.

Beginning in 1985, relatively penicillin G-resistant (Penr) meningococci which did not produce beta-lactamase were isolated from the blood and cerebrospinal fluid of patients in Spain. We identified 16 Penr (mean MIC, 0.3 microgram/ml; range, 0.1 to 0.7 microgram/ml) and 12 penicillin-susceptible (Pens; mean MIC, less than or equal to 0.06 microgram/ml) strains of Neisseria meningitidis by the agar dilution technique using an inoculum of 10(4) CFU and questioned which disk susceptibility test would best differentiate these two populations. We compared the disk susceptibility of these strains using disks containing 2 (P2) and 10 (P10) U of penicillin G, 2 (Am2) and 10 (Am10) micrograms of ampicillin, and 1 microgram of oxacillin (OX1). We also investigated susceptibility with disks containing 30 micrograms of each of cephalothin (CF30), cefoxitin (FOX30), cefuroxime (CXM30), and cefotaxime (CTX30) and 75 micrograms of cefoperazone (CFP75) and determined by cluster analysis any correlation with the zone diameters obtained with P2 disks. Using the P2 and AM2 disks (in contrast to the P10 and AM10 disks), we correctly differentiated all the Penr from Pens isolates. In addition, the zone diameters with the P2 disk gave the best correlation with the penicillin G MIC determinations. All 16 Penr strains and 3 of 12 Pens strains showed zone diameters of 6 mm around OX1 disks, limiting the usefulness of OX1 disks. The zone diameters obtained with CF30, CXM30, and OX1 disks correlated with those obtained with the P2 disk, which suggests that these antibiotics have similar effects on these strains. In contrast, the data obtained with FOX30, CTX30, and CFP75 disks did not cluster with those obtained with the P2 disk, which suggests that there was a difference in the bacterial target or reflects their greater activity. We conclude that the P2 disk tests more readily identify Penr meningococci than do the standard P10 disk tests.

Ampicillin resistance and penicillin-binding proteins of Haemophilus influenzae.

Penicillin-binding protein (PBP) alterations have been associated with non-beta-lactamase-mediated ampicillin resistance in Haemophilus influenzae. We evaluated the PBP profiles of several ampicillin-susceptible and -resistant clinical isolates of H. influenzae to determine how consistently the described alterations occurred, and to document the reproducibility of the PBP profiles for this species. The MIC of ampicillin ranged from 0.06 to 0.13 microgram ml-1 for the susceptible isolates at an inoculum of 100,000 c.f.u. when tested by broth dilution, and was 0.5 microgram ml-1 for all four isolates when tested by agar dilution. The MIC for the resistant isolates ranged from 4 to 8 micrograms ml-1 when tested by broth dilution, and from 1.5 to 16 micrograms ml-1 when tested by agar dilution. At least eight distinct PBPs with molecular masses ranging from 27 to 90 kDa were detected both in cell membrane preparations and whole cell (in vivo) binding assays done on cells in the exponential growth phase. PBP variability was evident both in the ampicillin-susceptible and -resistant isolates; however, much greater variability existed within the four resistant strains. The differences in PBP patterns included (1) electrophoretic mobility, (2) binding capacity for the antibiotic and (3) the presence of additional PBPs in two of the resistant isolates. However, decreased binding capacity was consistently demonstrated in PBP 5 (56 kDa) of all of the resistant isolates. Saturation curves with both penicillin and ampicillin indicated that PBP 5 had decreased affinity for the antibiotics.(ABSTRACT TRUNCATED AT 250 WORDS)

Failure to detect ampicillin-resistant, non-beta-lactamase-producing Haemophilus influenzae by standard disk susceptibility testing.

We questioned whether the apparent rarity of ampicillin-resistant, non-beta-lactamase-producing (NBLP) Haemophilus influenzae was due to failure of detection. We identified ampicillin-resistant and ampicillin-susceptible H. influenzae by the agar dilution technique, using 10(5)- and 10(3)-CFU inocula. We compared the disk susceptibility of 18 ampicillin-resistant NBLP strains, 13 ampicillin-resistant, beta-lactamase-producing strains, and 10 ampicillin-susceptible strains by using standard 10- and 2-micrograms ampicillin disks on two different media. We also investigated the possibility that disks containing 10 micrograms of clavulanic acid and 2 micrograms of ampicillin could be used to distinguish between the two ampicillin-resistant populations. Using the disk containing 2 micrograms of ampicillin, we correctly differentiated all the ampicillin-resistant isolates from the ampicillin-susceptible isolates on both media (a zone diameter of less than or equal to 15 mm was considered resistant). In contrast, the 10-micrograms ampicillin disk failed to detect 44% (8 of 18) of the ampicillin-resistant NBLP strains (a zone diameter of less than or equal to 19 mm was considered resistant). The MIC of ampicillin with the 10(3)-CFU inoculum correlated better with zone diameters than with the 10(5)-CFU inoculum. A difference in zone diameters of greater than or equal to 8 mm between the disk containing 10 micrograms of clavulanic acid and 2 micrograms of ampicillin and the disk containing only 2 micrograms of ampicillin correctly identified all beta-lactamase-producing strains. We conclude that the 2-micrograms ampicillin disk tests more readily identify ampicillin resistance in H. influenzae than do the 10-micrograms ampicillin disk tests. Future investigation should determine whether this in vitro resistance correlates with clinical treatment failures.

Characterization of non-beta-lactamase-mediated ampicillin resistance in Haemophilus influenzae.

Ampicillin resistance in Haemophilus influenzae is most often due to the plasmid-mediated production of TEM beta-lactamase. We studied four strains with high-level ampicillin resistance (MIC of 32 micrograms/ml with an inoculum of 10(5) CFU on solid media) which did not produce detectable beta-lactamase activity with two different detection methods. Two of the four strains contained extrachromosomal DNA by agarose gel electrophoresis. Conjugation failed to transfer ampicillin resistance; in contrast, transformation yielded ampicillin-resistant transformants in three of the four strains. These transformants did not contain detectable extrachromosomal DNA. In addition, mobilization of the resistance determinant by transformation to, or conjugation with, recombination-deficient strains was unsuccessful. DNA-DNA hybridization experiments revealed no homology of the DNA of these strains with two R plasmids (one coding for ampicillin resistance, the other for chloramphenicol and tetracycline resistance). We conclude that the genetic basis of the non-beta-lactamase ampicillin resistance in these strains appears to be chromosomally mediated. We investigated the mechanism of resistance in these strains. Enzymatic modification of penicillin was not detected by autoradiography of a thin-layer chromatogram of cell sonic extracts of three ampicillin-resistant transformant strains incubated with [14C]penicillin. To assess changes in permeability of the cell envelope, a plasmid coding for beta-lactamase was conjugated into these strains, and the hydrolysis of penicillin by intact cells and cell sonic extracts was compared. Only one of three transformant strains had significantly diminished permeability. Outer membrane proteins of these strains analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed apparent differences in comparison with the isogenic ampicillin-susceptible recipient strain. Autofluorography of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Sarkosyl-solubilized crude membrane (the putative inner membranes) from these ampicillin-resistant transformant strains incubated with [3H]penicillin compared with the isogenic ampicillin-susceptible recipient strain revealed reduced binding to PBP 3 and 6, 3 and 4, or 4. In addition, affinity binding studies revealed decreased affinity of PBP 4 for ampicillin of all four transformants tested. We conclude that the major mechanism of resistance in these strains is altered penicillin-binding proteins; however, other mechanisms, including permeability, may also play a role.