A mutation (env-10) conferring increased susceptibility to drugs, dyes, and detergents was detected in a clinical isolate of Neisseria gonorrhoeae. In certain strains, env-10 also affected susceptibility to pyocins. This mutation was phenotypically similar to but genotypically distinct from previously described env mutations.Isogenic strains of Neisseria gonorrhoeae containing mutations which confer either increased resistance or susceptibility to dyes, detergents, and drugs have been studied extensively in this laboratory (1,3,4,7). Using isogenic strains, Guymon et al. (3) demonstrated that the presence of an mtr (multiple transformable resistance) locus results in reduced outer membrane permeability and increased resistance to multiple antimicrobial agents. However, certain mutations, designated env, phenotypically reverse the effects of mtr (7) and result in increased outer membrane permeability and susceptibility to drugs, dyes, and detergents (3, 7). Two phenotypically identical but genetically distinct loci (env-J and env-3) independently suppress the effects of mtr (7). The env-J locus and the genetically indistinguishable env-2 locus result in decreased peptidoglycan cross-linking (4).Recently, we examined clinical isolates obtained from Denmark. One of us (I.L.) observed that one particularly resistant isolate (strain FA952) lost resistance to a variety of antimicrobial agents after several passages in vitro; a susceptible derivative of strain FA952 is designated strain FA953. We were curious to know whether loss of resistance was due to an env-like mutation. These and other gonococcal strains employed in this study are listed in Table 1.We determined the MICs of a variety of dyes, detergents, and antibiotics for selected strains (Table 2). We also examined the susceptibility of these strains toa pyocins (2, 6) and pooled normal human serum (8). Strain FA953 was remarkably susceptible to dyes, detergents, and antibiotics to which FA952 was relatively resistant; the phenotype of strain FA953 was similar to those of well-characterized isogenic env-], env-2, and env-3 strains BR54, BR84, and BR87. (7). Curiously, strains FA952 and FA953 differed from each other in susceptibility to lipopolysaccharide (LPS)-specific pyocins (2, 6): strain FA952 was susceptible to pyocins 1 and 103, whereas strain FA953 was resistant to the same pyocins.Results from three experiments suggested that strain FA953 contained an env mutation responsible for hypersusceptibility to the antimicrobial agents listed in Table 2. First, spontaneous erythromycin-resistant (Eryr) mutants of strain FA953 (e.g., strain FA968), which arose at a frequency of ca. * Corresponding author. t Present address: Department of Microbiology, Emory University School of Medicine, Atlanta, GA 30322.1.5 x 10-9, exhibited levels of multiple antimicrobial resistance similar to that of strain FA952. Second, strain FA953 Eryr transformants (e.g., strain FA970) obtained with sheared DNA from strain FA952 were also phenotypically identical to strain FA952. Thi...

The cell envelopes of antibiotic-resistant and -sensitive isogenic strains of Neisseria gonorrhoeae were analyzed to determine whether acquisition of genetic loci for altered antibiotic sensitivity was accompanied by alterations in cell envelope composition. No differences in the composition of phospholipids and lipopolysaccharides were noted. Acquisition of mtr-2, which results in low-level, nonspecific increased resistance to multiple antibiotics, dyes, and detergents, was accompanied by a sevenfold increase in the amount of a minor, 52,000-molecularweight outer membrane protein and a 32% increase in the extent of peptidoglycan cross-linking. Subsequent addition of the nonspecific hypersensitivity loci env-1 or env-2 to a strain carrying mtr-2 resulted in reversal of the phenotypic resistance determined by mtr-2 and marked reduction in both the amount of the 52,000molecular-weight outer membrane protein and the extent of peptidoglycan crosslinking. Introduction of penB2, which results in a fourfold increase in resistance to penicillin and tetracycline, was accompanied by the disappearance of the principal outer membrane protein of the wild-type strain (molecular weight, 36,900) and the appearance of a new species of the principal outer membrane protein (molecular weight, 39,400) in the transformant. MATERIALS AND METHODSStrains used. The strains used in these studies 391 on July 16, 2020 by guest

A lipopolysaccharide (LPS) mutant (FA5100) of a serum-resistant strain of Neisseria gonorrhoeae (FA19) was found to be highly sensitive to the bactericidal activity of normal human serum (NHS). Both strain FA5100 and an unrelated serum-sensitive clinical isolate (F62) were killed by NHS via the classical complement pathway since killing required C2 and Ca++. However, the fact that only strain FA5100 was sensitive to human hypogammaglobulinemic and cord serum suggested that this strain might activate the classical complement pathway in the absence of antibody. Anticomplementary concentrations of LPS from strain FA5100 inhibited the bactericidal activity of NHS against either strain FA5100 or strain F62. However, concentrations of LPS from strain FA5100 that exhibited marginal anticomplementary behavior also inhibited the killing of strain F62 by NHS. The ability of LPS from strain FA5100 to inhibit the bactericidal activity of NHS against strain FA5100 and to activate complement was reduced by treatment with mild alkali. However, alkali-treated LPS from strain FA5100 still inhibited the bactericidal activity of NHS against strain F62.

Dark-colored colony types of Neisseria gonorrhoeae (T3 and dark variants of T1 and T2) had markedly increased amounts of an approximately 28,000-dalton outer membrane protein, as compared with light-colored colony types (T4 and light variants of T1 and T2). The presence of this protein appeared to be unrelated to piliation. The apparent molecular weight of this protein on sodium dodecyl sulfate-polyacrylamide gels varied, depending on methods used to solubilize envelope proteins. In view of the location of this protein on the outer membrane, this protein could be important to the pathogenicity or antigenicity of the organism as well as to colonial characteristics in vitro.

Glucose transport by Pseudomonas aeruginosa was studied. These studies were enhanced by the use of a mutant, strain PAO 57, which was unable to grow on glucose but which formed the inducible glucose transport system when grown in media containing glucose or other inducers such as 2-deoxy-D-glucose. Both PAO 57 and parental strain PAO transported glucose with an apparent Km of 7 ,uM. Free glucose was concentrated intracellularly by P. aeruginosa PAO 57 over 200fold above the external level. These data constitute direct evidence that glucose is transported via active transport by P. aeruginosa. Various experimental data clearly indicated that P. aeruginosa PAO transported methyl a-D-glucose (a-MeGlc) via the glucose transport system. The apparent Km of a-MeGlc transport was 7 mM which indicated a 1,000-fold lower affinity of the glucose transport system for a-MeGlc than for glucose. While only unchanged a-MeGlc was detected intracellularly in P. aeruginosa, a-MeGlc was actually concentrated intracellularly less than 2-fold over the external level. Membrane vesicles of P. aeruginosa PAO retained transport activity for gluconate. This solute was concentrated intravesicularly several-fold over the external level. A component of the glucose transport system is believed to have been lost during vesicle preparation since glucose per se was not transported. Instead; glucose was converted to gluconate by membrane-associated glucose dehydrogenase and gluconate was then transported into the vesicles. Although this may constitute an alternate system for glucose transport, it is not a necessary prerequisite for glucose transport by intact cells since P. aeruginosa PAO 57, which lacks glucose dehydrogenase, was able to transport glucose at a rate equal to the parental strain.Glucose is transported by Pseudomonas aeruginosa by a specifically inducible system (3, 10). This transport system is temperature dependent, it obeys saturation kinetics, and it is inhibited by uncouplers. Evidence also indicates that glucose is transported by P. aeruginosa as the free sugar and not by the phosphoenolpyruvate: hexose phosphotransferase system (3, 10, 11).