Selection and Characterization of Phage-Resistant Mutant Strains of Listeria monocytogenes Reveal Host Genes Linked to Phage Adsorption

Listeria-infecting phages are readily isolated from Listeria-containing environments, yet little is known about the selective forces they exert on their host. Here, we identified that two virulent phages, LP-048 and LP-125, adsorb to the surface of Listeria monocytogenes strain 10403S through different mechanisms. We isolated and sequenced, using whole-genome sequencing, 69 spontaneous mutant strains of 10403S that were resistant to either one or both phages. Mutations from 56 phage-resistant mutant strains with only a single mutation mapped to 10 genes representing five loci on the 10403S chromosome. An additional 12 mutant strains showed two mutations, and one mutant strain showed three mutations. Two of the loci, containing seven of the genes, accumulated the majority (n = 64) of the mutations. A representative mutant strain for each of the 10 genes was shown to resist phage infection through mechanisms of adsorption inhibition. Complementation of mutant strains with the associated wild-type allele was able to rescue phage susceptibility for 6 out of the 10 representative mutant strains. Wheat germ agglutinin, which specifically binds to N-acetylglucosamine, bound to 10403S and mutant strains resistant to LP-048 but did not bind to mutant strains resistant to only LP-125. We conclude that mutant strains resistant to only LP-125 lack terminal N-acetylglucosamine in their wall teichoic acid (WTA), whereas mutant strains resistant to both phages have disruptive mutations in their rhamnose biosynthesis operon but still possess N-acetylglucosamine in their WTA.     

Transposon-induced mutations in two loci of Listeria monocytogenes serotype 1/2a result in phage resistance and lack of N-acetylglucosamine in the teichoic acid of the cell wall.

Teichoic acid-associated N-acetylglucosamine and rhamnose have been shown to serve as phage receptors in Listeria monocytogenes serotype 1/2a. We generated and characterized two single-copy Tn916DeltaE mutants which were resistant to phage A118 and several other serotype 1/2a-specific phages. In one mutant the insertion was immediately upstream of the recently identified ptsHI locus, which encodes two proteins of the phosphoenolpyruvate-dependent carbohydrate uptake system, whereas in the other the insertion was immediately upstream of an operon whose most distal gene was clpC, involved in stress responses and virulence. Transduction experiments confirmed the association of the phage-resistant phenotype of these mutants with the transposon insertion. Phage A118 resistance of the mutants could be attributed to inability of the phage to adsorb onto the mutant cells, and biochemical analysis of cell wall composition showed that the teichoic acids of both mutants were deficient in N-acetylglucosamine. Rhamnose and other teichoic acid and cell wall components were not affected.     

Cell Wall Composition and Associated Properties of Methicillin-Resistant Staphylococcus aureus Strains

Methicillin-resistant (MR) Staphylococcus aureus strains have previously been reported to be deficient in surface negative charge; this has been correlated with methicillin resistance and ascribed to a deficiency of teichoic acid at the cell surface (A. W. Hill and A. M. James, Microbios 6:157-167, 1972). Teichoic acid was present in walls of MR organisms as revealed by appreciable phosphate levels and detection of ribitol residues. Phosphate levels in walls from five MR strains (0.54 to 0.77 mumol/mg of wall) were lower than in three unrelated methicillin-sensitive (MS) strains (0.86 to 1.0 mumol/mg of wall). However, two MS strains derived from two of the MR strains had wall phosphate levels very similar to those of the MR strains. No evidence for unusual wall polymers was found. Simple deficiency of wall teichoic acid does not result in methicillin resistance since an independently isolated teichoic acid-deficient strain (0.1 mumol of phosphate per mg of wall) was not methicillin resistant. In studies of biological properties possibly related to wall teichoic acid, it was discovered that walls isolated from MR organisms grown in the presence of methicillin autolyzed more rapidly than those isolated from organisms grown in the absence of the drug. Since methicillin resistance is enhanced by NaCl and suppressed by ethylenediaminetetraacetate, the effects of these compounds on autolysis of isolated walls were studied. NaCl (1.0 M) and ethylenediaminetetraacetate (1.0 mM) inhibited the autolysis of walls isolated from MR and MS strains. An MR strain bound phage 47, 52A, and 3A only slightly less well than their respective propagating strains.     

Cell surface characteristics of some phage-resistant strains of Lactobacillus casei

The cell surface characteristics of two types of PL-1 phage-resistant strains of Lactobacillus casei: K-12, which does not permit adsorption of the phage; and YIT 9021, which permits phage adsorption but not genome injection, were compared with those of the parent strain. There were no differences in the electron microscopic features of the cell surface. However, both the phage-resistant strains were more hydrophobic than the parent strain as judged by hydrocarbon affinity test. A colloid titration test showed that these mutant strains were less negatively charged than the parent one. They differed in the composition of the polysaccharide of their cell walls, which were all free from teichoic acids.     

Cell wall components in Staphylococcus aureus with double resistance to gramicidin S and actinomycin D]

Cell walls of Staphylococcus aureus R9/80 resistant to gramicidin S and actinomycin D were investigated. The strain was isolated after passages of a previously isolated strain of S. aureus with resistance to gramicidin and definite changes in the cell walls, a medium with increasing concentrations of actinomycin being used for the passages. The data on the study of the cell walls of the strain with the double resistance were compared with the results of the investigation of the cell walls of the strain susceptible to gramicidin, the gramicidin resistant strain (initial for strain R9/80) and the actinomycin adapted strain that also showed changes in the cell walls. The cell walls of the resistant strains had no significant changes in the peptidoglycane and glucosamine levels, as well as in the peptidoglycane amino acid composition. Teichoic acids of all the strains had different levels of substitution of ribite by D-alanine (a factor influencing the negative charge of teichoic acids and the wall at large). It was noted that all the strains resistant to the tested antibiotics had lower levels of teichoic acids in the cell walls. The resistant cells showed some increase of the lipid component in the walls: from 1.6% in the susceptible strain to 2.1-2.9% in the resistant cells. The main trend of the changes in the resistance development was revealed to be the thickening of the cell wall and its consolidation. The development of resistance to gramicidin, actinomycin and to both the antibiotics provoked respectively a 2.4-, 4- and 5.4-fold increase of the content of the main cell component. i.e. peptidoglycane in the cell biomass. The barrier role of the cell walls in the resistant strains and their ability to bind the antibiotic is discussed.     

Transposon-Induced Mutations in Two Loci of Listeria monocytogenes Serotype 1/2a Result in Phage Resistance and Lack of N-Acetylglucosamine in the Teichoic Acid of the Cell Wall

Teichoic acid-associated N-acetylglucosamine and rhamnose have been shown to serve as phage receptors in Listeria monocytogenes serotype 1/2a. We generated and characterized two single-copy Tn916ΔE mutants which were resistant to phage A118 and several other serotype 1/2a-specific phages. In one mutant the insertion was immediately upstream of the recently identified ptsHI locus, which encodes two proteins of the phosphoenolpyruvate-dependent carbohydrate uptake system, whereas in the other the insertion was immediately upstream of an operon whose most distal gene was clpC, involved in stress responses and virulence. Transduction experiments confirmed the association of the phage-resistant phenotype of these mutants with the transposon insertion. Phage A118 resistance of the mutants could be attributed to inability of the phage to adsorb onto the mutant cells, and biochemical analysis of cell wall composition showed that the teichoic acids of both mutants were deficient in N-acetylglucosamine. Rhamnose and other teichoic acid and cell wall components were not affected.     

Function of cell wall teichoic acid in thermally injured Staphylococcus aureus.

Thermally injured cells of Staphylococcus aureus lack the ability to grow on tryptic soy agar containing 7.5% NaCl. This injury phenomenon was examined in three strains of S. aureus: MF-31; H (Str); and, isolated from H (Str), 52A5, a mutant which lacks teichoic acid in the cell wall. Temperatures for sublethal heat treatment were selected to produce maximum injury with minimum death for each strain. Examination of isolated cell walls showed that magnesium was lost from the wall during heating, and that the degree of cell injury was accentuated when magnesium ions were either removed from or made unavailable to the cell. S. aureus 52A5 was more heat sensitive than its parent strain. Cells containing higher levels of wall teichoic acid generally showed less injury than normal cells. Cells with the weaker cation-binding polymer, teichuronic acid, in the cell wall generally showed greater injury. These data suggest that cell wall teichoic acid of S. aureus aids in the survival of the cell by the maintenance of an accessible surface pool of magnesium.     

Relation between wall teichoic acid content of Bacillus subtilis and efficiency of adsorption of bacteriophages SP 50 and Φ 25

Efficient adsorption of bacteriophages SP 50 and 25 occurred only to bacilli that contained wall teichoic acid and neither phage bound to phosphate limited bacilli that contained teichuronic acid instead of teichoic acid. Though both phages require the presence of teichoic acid, their receptors are not identical. Efficient binding of phage 25 required the presence of greater proportions of teichoic acid in the wall and the receptor for this phage was destroyed when bacteria or isolated walls were heated at pH 4 whereas the ability of these samples to bind phage SP 50 was unaffected by such treatment. Efficient binding of phage SP 50 was not highly dependant on the presence of glucosyl substituents on the teichoic acid. Such substituents were required for phage 25 binding though their anomeric configuration appeared to be unimportant since the phages bound well to both strains W23 and 168, the wall teichoic acids of which carry glucosyl substituents of opposite anomeric configuration. The differeneces in the nature of the receptors may be of value in the use of the phages as probes for the location and distribution of teichoic acid in the wall.Non-Standard Abbreviation PAE Phage adsorption efficiency, as defined by Archibald and Coapes (1976)     

Investigation of cell wall components in actinomycin D resistant Staphylococcus aureus]

Cell walls in 2 strains of Staphylococcus aureus 209P, i.e. actinomycin D susceptible and resistant ones were comparatively investigated. The resistant cells contained much more wall material per a unit of the biomass weight vs the susceptible strain cells, that conformed to thickening of the resistant cell walls detected by electron microscopy and a sharp increase of their electron density. Investigation of peptidoglycans and teichoic acids did not reveal any significant alterations in the structure of the wall components in the actinomycin D resistant cells. Only some increase of glucosamine in the peptidoglycan fraction of the resistant cells vs the susceptible ones was observed. It was shown that preparations of the resistant cell walls and peptidoglycan isolated from the resistant cells were able to bind somewhat lower quantities of actinomycin D vs the analogous preparations of the susceptible cells. The significant decrease of the antibiotic binding by live cells of the resistant strain probably slightly depended on the structure characteristics of the main wall components. The barrier properties of the walls in resistant staphylococci are most likely defined by the wall thickening and consolidation while adapting to actinomycin D.     

Cell wall components of gramicidin S resistant Staphylococcus aureus]

Comparative study of two staphylococcus aureus 209P strains--resistant and susceptible to gramicidin S demonstrated that peptidoglycanes of two strains differ by ratio glycine/serine at peptide bridges. Besides peptidoglycanes significantly differ by amidation of alfa-carboxyles of glutamic acid in muropeptide. This peptidoglycane modification of resistant cells along with enhanced content of etherized D-alanine in teichoic acid provides lower negative charge of cell wall components. It may influence the cell wall ability to react with positively charged gramicidin molecules. It was shown that isolated cell walls and peptidoglycane of resistant cells binds significantly less gramicidin than cell walls and peptodoglyce of susceptable cells. Simultaneous determination of gramicidin binding by intact S. aureus cells and their killing revealed that lower ability of resistant cells to bind gramicidin is significant but not critical factor of gramicidin resistance.     

Properties of a Novel Pleiotropic Bacteriophage-Resistant Mutant of Staphylococcus aureus H

A phage-resistant mutant of Staphylococcus aureus H (Sm(R)), S. aureus 52A5, was previously shown to lack polymeric teichoic acid. This paper characterizes other phenotypic differences between the strains. In broth cultures the mutant cells grew more slowly, were larger, and formed much larger clumps than the parent strain. The clumps of cells appeared to be covalently linked and could only be separated by mild sonic energy-a process which yielded viable cells. Mutant and parent cells autolyzed at equal rates, whereas isolated cell walls of the mutant strain autolyzed faster than the wild type. Nevertheless, the specific activity of the autolytic enzyme in the wild type soluble fraction was much higher than in the mutant. In contrast to the parent, strain 52A5 failed to accumulate nucleotide-bound murein precursors when treated with penicillin. Mutant strains with these characteristics were repeatedly isolated both spontaneously and by chemical mutagenesis. Strain 52A5 was shown to be fully revertible. Thus, it appears to be a pleiotropic mutation, and the possible nature of the defect which causes these varied effects is discussed.     

Chemical characterization of a new surface antigenic polysaccharide from a mutant of Staphylococcus aureus

A mutant of Staphylococcus aureus H was isolated by virtue of its inability to agglutinate with antibodies against teichoic acid of S. aureus. Immunological studies revealed that the mutant, S. aureus T, possessed a new surface antigen in addition to having the antigenic determinant of the wild-type strain, the ribitol teichoic acid. The presence of this additional surface component rendered strain T resistant to staphylococcal typing phages, presumably by masking the phage-receptor sites. The polymer was separated from teichoic acid by chromatography on diethylaminoethyl cellulose and was shown to be composed of two amino sugars, N-acetyl-d-fucosamine and N-acetyl-d-mannosamin uronic acid.     

Choline-containing bacteriophage receptors in Streptococcus pneumoniae.

Choline-containing teichoic acid seems to be essential for the adsorption of bacteriophage Dp-1 to pneumococci. This conclusion is based on the following observations: In contrast to pneumococci grown in choline-containing medium, cells grown in medium containing ethanolamine or other submethylated aminoalcohols instead of choline were found to be resistant to infection by Dp-1. Live choline-grown bacteria and heat- or UV-inactivated cells and purified cell walls prepared from these cells were capable of adsorbing phage Dp-1; ethanolamine-grown pneumococci or cell wall preparations were unable to do so. Adsorption of Dp-1 to choline-containing cell walls was competitively inhibited by phosphorylcholine and by several choline-containing soluble cell surface components, such as the Forssman antigen and the teichoic acid-glycan complexes formed by autolytic cell wall degradation. Cell walls prepared from pneumococci grown in ethanolamine or phosphorylethanolamine were inactive. Electron microscopic studies with pneumococci that had segments of choline-containing cell wall material amid ethanolamine-containing regions indicated that the Dp-1 phage particles adsorbed exclusively to the choline-containing surface areas. We suggest that the choline residues of the pneumococcal teichoic acid are essential components of the Dp-1 phage receptors in this bacterium.     

Lysogenic conversion for multiple characters in a strain of Staphylococcus aureus.

Lysogenization of nonlysogenic strains of Staphylococcus aureus was performed with two different bacteriophages, LS1 and LS2, that were unable to plaque on any of the strains of S. aureus tested. Infection of recipient strains was achieved when protoplasts were inoculated with LS1 or LS2 or when bacterial cultures were simultaneously inoculated with a virulent phage together with LS1 or LS2. Lysogenization was demonstrated by changes in phenotypic characters of the host strain and by liberation of bacteriophages from the modified strains as shown by electron microscopic examination. The lysogenic strains differed from the host strains by the following characters: they were coagulase, deoxyribonuclease, and lipase negative; they were untypable by the basic set of phages; they did not ferment mannitol under anaerobic conditions; and they produced only l-(+)-lactic acid by glucose fermentation. Their cell walls contained less glycine and concomitantly more serine than those of the host strains. Furthermore, they were devoid of protein A. Conversely, some antigenic factors as well as the presence of ribitol in the cell wall teichoic acid, indicated a parental relationship between the host strains and the derived lysogenic ones. Phages LS1 and LS2 could be excluded from the lysogenic strains by invading phages, and the revertant nonlysogenic strains recovered all of the characteristics of the initial host strains. It was thus concluded that the phenomenon described was due to lysogenic conversion. The origin of phages LS1 and LS2 is discussed.     

A mutant bacteriophage evolved to infect resistant bacteria gained a broader host range

Bacteriophages (phages) are the most abundant entities in nature, yet little is known about their capacity to acquire new hosts and invade new niches. By exploiting the Gram‐positive soil bacterium Bacillus subtilis (B. subtilis) and its lytic phage SPO1 as a model, we followed the coevolution of bacteria and phages. After infection, phage‐resistant bacteria were readily isolated. These bacteria were defective in production of glycosylated wall teichoic acid (WTA) polymers that served as SPO1 receptor. Subsequently, a SPO1 mutant phage that could infect the resistant bacteria evolved. The emerging phage contained mutations in two genes, encoding the baseplate and fibers required for host attachment. Remarkably, the mutant phage gained the capacity to infect non‐host Bacillus species that are not infected by the wild‐type phage. We provide evidence that the evolved phage lost its dependency on the species‐specific glycosylation pattern of WTA polymers. Instead, the mutant phage gained the capacity to directly adhere to the WTA backbone, conserved among different species, thereby crossing the species barrier.     

Regulation of polar surface structures in Caulobacter crescentus: Pleiotropic mutations affect the coordinate morphogenesis of flagella, pili and phage receptors

Summary A large number of Caulobacter mutants resistant to DNA or RNA phages were isolated. These phage-resistant mutants exhibited phenotypic variations with respect to cell motility and sensitivity to other phages.The majority of the mutants was resistant to both DNA and RNA phages tested. In addition, these mutants were either motile or non-motile. The analysis of spontaneous revertants from these mutants indicated that a single mutation is involved in these phenotypic variations. Other mutants were resistant to RNA phages and only to a certain DNA phage tested, and were also motile or non-motile.Several temperature-sensitive phage-resistant mutants were also isolated. One of them, CB13 ple-801, exhibited the wild type phenotype when grown at 25°C. However, at a higher temperature (35°C), the mutant cells became non-motile and resistant to both DNA and RNA phages. These phenotypes seem to be attributed to the concommitant loss of flagella, pili and phage receptors. In other respects (cell growth and morphology, and asymmetric stalk formation), CB13 ple-801 was normal at 35°C. The spontaneous revertants from CB13 ple-801 simultaneously regained the wild type phenotypes in all respects.It is suggested that a single mutation pleiotropically affects the formation of flagella, pili and phage receptors.     

Isolation and analysis of cell wall components from Streptococcus pneumoniae

The complex and heterogeneous cell wall of the pathogenic bacterium Streptococcus pneumoniae is composed of peptidoglycan and a covalently attached wall teichoic acid. The net-like peptidoglycan is formed by glycan chains that are crosslinked by short peptides. We have developed a method to purify the glycan chains, and we show that they are longer than approximately 25 disaccharide units. From purified peptidoglycan, we released 50 muropeptides that differ in the length of their peptides (tri-, tetra-, or pentapeptides with or without mono- or dipeptide branch), the degree of peptide crosslinking (monomer, dimer, or trimer), and the presence of modifications in the glycan chains (N-deacetylation, O-acetylation, or lack of GlcNAc or GlcNAc-MurNAc) or peptides (glutamic acid instead of glutamine). We also established a method to isolate wall teichoic acid chains and show that the most abundant chains have 6 or 7 repeating units. Finally, we obtained solid-state nuclear magnetic resonance spectra of whole insoluble cell walls. These novel tools will help to characterize mutant strains, cell wall-modifying enzymes, and protein-cell wall interactions.     

Regulation of bacterial cell walls: correlation between autolytic activity and cell wall turnover in Staphylococcus aureus.

Cell wall turnover was examined in parent and mutant strains of Staphylococcus aureus. Peptidoglycan and teichoic acid were observed to undergo turnover in the wild-type strain during exponential growth; however, the rate of turnover did not decrease when the growth rate slowed, as the culture entered stationary phase. Isolated native cell walls and crude soluble autolytic enzyme were prepared from cells harvested during exponential and postexponential phases of growth. Native cell walls from both phases of growth autolyzed in buffer at identical rates; similarily, crude soluble enzyme from both preparations degraded radioactive cell walls at the same rate. Therefore, the activity of the autolysin in both exponential and postexponential cells was similar. The autolysis of whole cells of a mutant tar-1 was enhanced by 1.0 M NaCl. When 1.0 M NaCl was present under growing conditions, the rate of cell wall turnover was greatly increased. The presence of chloramphenicol, which inhibits whole-cell autolysis, also inhibited turnover. Analysis of the cell wall material recovered from spent medium revealed products consistent with the known mode of action of the endogenous autolysin. It is concluded that cell wall turnover in S. aureus is independent of the stage of culture growth but is dependent instead on the activity of the autolysin.     

The cell wall binding domain of Listeria bacteriophage endolysin PlyP35 recognizes terminal GlcNAc residues in cell wall teichoic acid

The cell wall binding domains (CBD) of bacteriophage endolysins target the enzymes to their substrate in the bacterial peptidoglycan with extraordinary specificity. Despite strong interest in these enzymes as novel antimicrobials, little is known regarding their interaction with the bacterial wall and their binding ligands. We investigated the interaction of Listeria phage endolysin PlyP35 with carbohydrate residues present in the teichoic acid polymers on the peptidoglycan. Biochemical and genetic analyses revealed that CBD of PlyP35 specifically recognizes the N-acetylglucosamine (GlcNAc) residue at position C4 of the polyribitol-phosphate subunits. Binding of CBDP35 could be prevented by removal of wall teichoic acid (WTA) polymers from cell walls, and inhibited by addition of purified WTAs or acetylated saccharides. We show that Listeria monocytogenes genes lmo2549 and lmo2550 are required for decoration of WTAs with GlcNAc. Inactivation of either gene resulted in a lack of GlcNAc glycosylation, and the mutants failed to bind CBDP35. We also report that the GlcNAc-deficient phenotype of L. monocytogenes strain WSLC 1442 is due to a small deletion in lmo2550, resulting in synthesis of a truncated gene product responsible for the glycosylation defect. Complementation with lmo2550 completely restored display of characteristic serovar 1/2 specific WTA and the wild-type phenotype.     

Host ranges of Listeria-specific bacteriophages from the turkey processing plant environment in the United States

Even though at least 400 Listeria phages have been isolated from various sources, limited information is available on phages from the food processing plant environment. Phages in the processing plant environment may play critical roles in determining the Listeria population that becomes established in the plant. In this study, we pursued the isolation of Listeria-specific phages from environmental samples from four turkey processing plants in the United States. These environmental samples were also utilized to isolate Listeria spp. Twelve phages were isolated and classified into three groups in terms of their host range. Of these, nine (group 1) showed a wide host range, including multiple serotypes of Listeria monocytogenes, as well as other Listeria spp. (L. innocua, L. welshimeri, L. seeligeri, and L. ivanovii). The remaining phages mostly infected L. monocytogenes serotype 4b as well as L. innocua, L. ivanovii, and/or L. welshimeri. All but one of the strains of the serotype 4b complex (4b, 4d, 4e) from the processing plant environment could be readily infected by the wide-host-range phages isolated from the environment of the processing plants. However, many strains of other serotypes (1/2a [or 3a] and 1/2b [or 3b]), which represented the majority of L. monocytogenes strains isolated from the environmental samples, were resistant to infection by these phages. Experiments with two phage-resistant strains showed reduced phage adsorption onto the host cells. These findings suggest that phage resistance may be an important component of the ecology of L. monocytogenes in the turkey processing plants.