Restriction and Modification in Group N Streptococci: Effect of Heat on Development of Modified Lytic Bacteriophage

The appearance of lytic bacteriophage against newly introduced starter strains used during commercial cheese manufacture occurs rapidly, and their origin is not well understood. In this study, members of the group N streptococci were examined for the presence of bacteriophage restriction and modification systems. Two streptococcal phages from Streptococcus cremoris TR and Streptococcus lactis C2 (phage designations tr and c2) showed restricted lytic development on S. cremoris 799 and KH, respectively. Efficiency of plaquing was 1.9 × 10⁻⁷ for tr plaqued on 799 and 2.1 × 10⁻⁷ for c2 plaqued on KH. After passage through the restrictive hosts, these phages demonstrated high lytic ability for formerly restrictive hosts. Stress of the restrictive host strains at temperatures of 40 to 50°C resulted in a significant increase in the efficiency of plaquing of restricted bacteriophages. Elevated temperatures are encountered during commercial cheese manufacture. The results suggested that the temporary loss of host restriction activity with the resulting modification of nonspecific bacteriophage may contribute directly to the appearance of lytic phage against new starter strains.     

Conjugal Transfer of Bacteriophage Resistance Determinants on pTR2030 into Streptococcus cremoris Strains

Agar surface conjugal matings were used to introduce heat-sensitive phage resistance (Hsp⁺) determinants carried on the conjugal plasmid pTR2030 into Streptococcus cremoris KH, HP, 924, and TDM1. Lactose-fermenting (Lac⁺) transconjugants were selected from matings of Lac⁻ variants of S. cremoris KH, HP, 924, and TDM1 with Streptococcus lactis ME2 or a high-frequency donor, S. lactis T-EK1 (pTR1040, Lac⁺; pTR2030, Hsp⁺). For all of the S. cremoris strains examined, select Lac⁺ transconjugants were completely resistant to plaquing by their homologous lytic phages. In all cases the plaquing efficiencies were less than 10⁻⁹. Acquisition of a 30-megadalton plasmid (pTR2030) in the S. cremoris phage-resistant transconjugants was demonstrated by direct plasmid analysis, by hybridization with ³²P-labeled probes, or by conjugal transfer of pTR2030 out of the phage-resistant transconjugants into a plasmid-cured recipient, S. lactis LM2302. Acid production, coagulation ability, and proteolytic activity of phage-resistant transconjugants in milk were comparable to those of their phage-sensitive parents. Further, S. cremoris phage-resistant transconjugants were not attacked by phage in starter culture activity tests, which included a 40°C incubation period. The results demonstrated that phage resistance determinants on pTR2030 could be conjugally transferred to a variety of S. cremoris strains and confer resistance to phage under conditions encountered during cheese manufacture. Phage-resistant transconjugants of S. cremoris M43 and HP were also constructed without the use of antiblotic markers to select conjugal recipients from mating mixtures.     

Characterization of Phage-Sensitive Mutants from a Phage-Insensitive Strain of Streptococcus lactis: Evidence for a Plasmid Determinant that Prevents Phage Adsorption

A phage-insensitive strain of Streptococcus lactis, designated ME2, was used as a prototype strain for the study of mechanisms and genetics of phage resistance in the lactic streptococci. Mutants sensitive to a Streptococcus cremoris phage, ϕ18, were isolated at a level of 17% from cultures of ME2 after sequential transfer at 30°C. Phage-sensitive mutants of ME2 were not fully permissive to ϕ18. The efficiency of plating of ϕ18 on the mutants was 5 × 10⁻⁷ as compared with <10⁻⁹ for ϕ18 on ME2. Further characterization of the mutants showed that they efficiently adsorbed ϕ18 at levels of >99.8%, whereas ME2 adsorbed only 20 to 40% of ϕ18. These results suggest that increased phage susceptibility of the mutants may result from the loss of a mechanism that inhibits phage adsorption. Moreover, the high frequency of spontaneous mutation in ME2 indicates the involvement of an unstable genetic determinant in this phage defense mechanism. ME2 was shown to possess 13 plasmids ranging in size from 1.6 to 34 megadaltons. Of 40 mutants examined that had increased efficiencies of plating, all were missing a 30-megadalton plasmid, pME0030. These data suggest that pME0030 codes for a function that prevents phage adsorption. Further phenotypic characterization of the phage-sensitive mutants showed that some mutants were deficient in the ability to ferment lactose (Lac) and hydrolyze milk proteins (Prt). However, the Lac⁺ and Prt⁺ phenotype segregated independently of the phage-sensitivity phenotype. One phage-sensitive adsorption mutant, designated N1, was tested for susceptibility to 14 different phages. N1 showed increased capacity to adsorb 4 and to replicate 2 of these 14 phages, thereby indicating a phage resistance mechanism in ME2 that generalizes to phage interactions other than the specific ϕ18-ME2 phage-host interaction. These data provide evidence for a unique plasmid-linked phage defense mechanism in phage-insensitive strains of lactic streptococci.     

A Simple and Rapid Method for Genetic Transformation of Lactic Streptococci by Electroporation

An electroporation procedure for the plasmid-mediated genetic transformation of intact cells of Streptococcus cremoris and Streptococcus lactis was performed. Ten different strains were transformed. The method was simple and rapid and yielded transformant colonies in 14 to 24 h. The method was optimized for S. lactis LM0230, and transformation frequencies of between 1 × 10⁴ and 5 × 10⁵ transformants per μg of purified plasmid (pMU1328) were achieved routinely. The optimized procedure involved lysozyme treatment of cells. Transformation of LM0230 occurred at comparable frequencies with pLS1 (4.4 kilobase pair [kbp]), pMU1328 (7.4 kbp), and pAMβ1 (26.5 kbp). Plasmid DNA isolated from transformants had not undergone detectable deletions or rearrangements. Transformation was possible with plasmid DNA which was religated after restriction endonuclease digestion. Phage DNA-dependent transfection of S. lactis LM0230 and S. lactis C6 was also achieved.     

Plasmid-Determined Systems for Restriction and Modification Activity and Abortive Infection in Streptococcus cremoris

Streptococcus cremoris strain IL964 possessed a restriction and modification (R/M) activity which resulted in a bacteriophage efficiency of plating of 5 × 10⁻⁶. Phage sensitivity of protoplast-induced plasmid-cured derivatives indicated that two plasmids called pIL103 (5.7 kilobases) and pIL107 (15.2 kilobases) were each coding for one R/M system. Plasmid pIL103-encoded R/M was ascertained by transfer into the plasmid-free, R⁻/M⁻ strain IL1403 of S. lactis, using protoplast cotransformation. This procedure failed for pIL107 because of some degree of incompatibility between pIL107 and the indicator plasmid pHV1301 used in cotransformation experiments. We also observed that plasmid pIL105 (8.7 kilobases) which showed no incidence on phage sensitivity in the parental strain IL964, mediated abortive infection in strain IL1403. In 97% of the infected cells, the phage infection was abortive, while in the remaining 3% phages were produced with a decreased burst size (50 instead of 180).     

Conjugal Transfer in Lactic Streptococci of Plasmid-Encoded Insensitivity to Prolate- and Small Isometric-Headed Bacteriophages

Eight of 40 strains of Streptococcus lactis and S. lactis subsp. diacetylactis were able to conjugally transfer a degree of phage insensitivity to Streptococcus lactis LM0230. Transconjugants from one donor strain, S. lactis subsp. diacetylactis 4942, contained a 106-kilobase (kb) cointegrate plasmid, pAJ1106. The plasmid was conjugative (Tra⁺) and conferred phage insensitivity (Hsp) and lactose-fermenting ability (Lac) in S. lactis and Streptococcus cremoris transconjugants. The phage resistance mechanism was effective against prolate- and small isometric-headed phages at 30°C. In S. lactis transconjugants, the phage resistance mechanism was considerably weakened at elevated temperatures. A series of deletion plasmids was isolated from transconjugants in S. cremoris 4854. Deletion plasmids were pAJ2074 (74 kb), Lac⁺, Hsp⁺, Tra⁺; pAJ3060 (60 kb), Lac⁺, Hsp⁺; and pAJ4013 (13 kb), Lac⁺. These plasmids should facilitate mapping Hsp and tra genes, with the aim of constructing phage-insensitive strains useful to the dairy industry.     

Transduction of Lactose Metabolism by Streptococcus cremoris C3 Temperate Phage

Temperate phage was induced from Streptococcus cremoris C3 and morphologically characterized by high-resolution electron micrographic techniques. Interspecies genetic transfer of lactose-fermenting ability by the temperate phage was demonstrated, using two lactose-negative (Lac⁻) S. lactis strains as recipients. Plasmid transfer was confirmed by agarose gel electrophoresis. Transductant plasmid profiles were of three types—those containing no visible plasmid deoxyribonucleic acid, those possessing a 23-megadalton (Mdal) plasmid, and those containing a 23-Mdal plasmid and a 30-Mdal plasmid. A Lac⁺ transductant could serve as a donor of the lac determinants during solid-surface matings. These results add to previously published reports of inter- and intraspecies genetic transfer in dairy starter cultures.     

Concomitant Conjugal Transfer of Reduced-Bacteriophage-Sensitivity Mechanisms with Lactose- and Sucrose-Fermenting Ability in Lactic Streptococci

Ten previously reported lactose-positive (Lac⁺) transconjugants from Streptococcus lactis, S. cremoris, and S. lactis subsp. diacetylactis and one sucrose-positive (Suc⁺) transconjugant from S. lactis were examined for their sensitivity to prolate- and small isometric-headed bacteriophages. Four of the Lac⁺ transconjugants showed a 10- to 100-fold reduction in the efficiency of plating (EOP) as well as a reduced plaque size for the prolate phage c2 and were insensitive to the small isometric phage 712. A fifth Lac⁺ transconjugant demonstrated a similar reduced sensitivity to phage c2; however, this transconjugant was able to plaque phage 712, but with a reduced plaque size and EOP. The other five Lac⁺ transconjugants were sensitive to both c2 and 712 phages. The Suc⁺ transconjugant plaqued phage 712 with a reduced plaque size and EOP, but no reduction in plaque size or EOP was observed for phage c2. The Lac⁺ and reduced bacteriophage sensitivity (Rbs⁺) phenotypes were correlated with specific plasmids in the Lac⁺ transconjugants. As four of the Lac⁺ transconjugants exhibited a phenotypically indistinguishable Rbs⁺, one (AB001) was selected for further study. The Rbs⁺ in AB001 for both small isometric- and prolate-headed phages was not related to adsorption, and the reduced EOP for phage c2 was not related to the presence of a restriction and modification system. The latent period for phage c2 was unchanged, but the burst size was reduced 80%. The presence of the plasmid coding for Rbs⁺ retarded the lysis of a mitomycin C-induced prophage-containing strain. The Rbs⁺ mechanism appears to be abortive phage infection. This study supports previous observations that Rbs⁺ and conjugal transfer ability are physically linked among some group N streptococci. The results presented have implications in the identification of plasmids coding for Rbs⁺ and may also aid in explaining the dissemination of Rbs⁺ genes among lactic streptococci.     

Bacteriophage Resistance Conferred on Lactic Streptococci by the Conjugative Plasmid pTR2030: Effects on Small Isometric-, Large Isometric-, and Prolate-Headed Phages

A series of reactions between phages, sensitive hosts, and transconjugants where the sensitivity of small isometric-, large isometric-, and prolate-headed phages to pTR2030-induced phage resistance was evaluated in Streptococcus lactis and Streptococcus cremoris strains. Phage-resistant transconjugants were constructed in the desired host by conjugal transfer of lactose-fermenting ability (Lac⁺, pTR1040) and phage resistance (Hsp⁺, pTR2030) from S. lactis TEK1. S. lactis and S. cremoris transconjugants harboring pTR2030 were resistant to all small isometric-headed phages examined. In contrast, prolate- and large isometric-headed phages were either not inhibited in the pTR2030 transconjugants or exhibited a reduction in plaque size without a reduction in the efficiency of plaquing. Small isometric-headed phages subject to pTR2030 induced inhibition shared no significant DNA homology with pTR2030, suggesting that phage immunity genes are not harbored on the plasmid or responsible for resistance. The general effectiveness of pTR2030 against small isometric-headed phages was highly significant since these are the phages which have been isolated most commonly from dairy fermentation plants.     

Application of Bacteriophages To Control Intestinal Escherichia coli O157:H7 Levels in Ruminants

A previously characterized O157-specific lytic bacteriophage KH1 and a newly isolated phage designated SH1 were tested, alone or in combination, for reducing intestinal Escherichia coli O157:H7 in animals. Oral treatment with phage KH1 did not reduce the intestinal E. coli O157:H7 in sheep. Phage SH1 formed clear and relatively larger plaques on lawns of all 12 E. coli O157:H7 isolates tested and had a broader host range than phage KH1, lysing O55:H6 and 18 of 120 non-O157 E. coli isolates tested. In vitro, mucin or bovine mucus did not inhibit bacterial lysis by phage SH1 or KH1. A phage treatment protocol was optimized using a mouse model of E. coli O157:H7 intestinal carriage. Oral treatment with SH1 or a mixture of SH1 and KH1 at phage/bacterium ratios ≥10² terminated the presence of fecal E. coli O157:H7 within 2 to 6 days after phage treatment. Untreated control mice remained culture positive for >10 days. To optimize bacterial carriage and phage delivery in cattle, E. coli O157:H7 was applied rectally to Holstein steers 7 days before the administration of 10¹⁰ PFU SH1 and KH1. Phages were applied directly to the rectoanal junction mucosa at phage/bacterium ratios calculated to be ≥10². In addition, phages were maintained at 10⁶ PFU/ml in the drinking water of the phage treatment group. This phage therapy reduced the average number of E. coli O157:H7 CFU among phage-treated steers compared to control steers (P < 0.05); however, it did not eliminate the bacteria from the majority of steers.     

Conjugal Transfer of Lactose-Fermenting Ability Among Streptococcus cremoris and Streptococcus lactis Strains

Streptococcus cremoris C3 was found to transfer lactose-fermenting ability to LM2301, a Streptococcus lactis C2 lactose-negative streptomycin-resistant (Lac⁻ Str^r) derivative which is devoid of plasmid deoxyribonucleic acid (DNA); to LM3302, a Lac⁻ erythromycin-resistant (Ery^r) derivative of S. lactis ML3; and to BC102, an S. cremoris B₁ Lac⁻ Ery^r derivative which is devoid of plasmid DNA. S. cremoris strains R1, EB₇, and Z8 were able to transfer lactose-fermenting ability to LM3302 in solid-surface matings. Transduction and transformation were ruled out as mechanisms of genetic transfer. Chloroform treatment of donor cells prevented the appearance of recombinant clones, indicating that viable cell-to-cell contact was responsible for genetic transfer. Transfer of plasmid DNA was confirmed by agarose gel electrophoresis. Transconjugants recovered from EB₇ and Z8 matings with LM3302 exhibited plasmid sizes not observed in the donor strains. Transconjugants recovered from R1, EB₇, and Z8 matings with LM3302 were able to donate lactose-fermenting ability at a high frequency to LM2301. In S. cremoris R1, EB₇, and Z8 matings with LM2301, streptomycin resistance was transferred from LM2301 to the S. cremoris strains. The results confirm genetic transfer resembling conjugation between S. cremoris and S. lactis strains and present presumptive evidence for plasmid linkage of lactose metabolism in S. cremoris.     

Plasmid Profiles of Lactose-Negative and Proteinase-Deficient Mutants of Streptococcus lactis C10, ML3, and M18

Lactose- and proteinase-negative (Lac⁻ Prt⁻) mutants of Streptococcus lactis C10, ML3, and M18 were isolated after treatment with ethidium bromide. The Lac⁻ Prt⁻ mutants of C10 were missing a 40-megadalton plasmid. A 33-megadalton plasmid was absent in the ML3 mutants, and the M18 variants lacked a 45-megadalton plasmid. The results suggest a linkage of these metabolic traits to the respective plasmids. The possible complexity of the interrelationship between lactose metabolism and proteinase activity is presented.     

Conjugal Strategy for Construction of Fast Acid-Producing, Bacteriophage-Resistant Lactic Streptococci for Use in Dairy Fermentations

Bacteriophage-resistant dairy streptococci were obtained following conjugal transfer of pTR2030 from a lactose-negative donor, Streptococcus lactis TEK12, to lactose-positive recipient strains, Streptococcus cremoris LMA13 and 924 and S. lactis LMA12. Fast acid-producing, phage-resistant transconjugants were selected by challenge with homologous phage on fast-slow differential agar or lactose indicator agar. Acquisition of pTR2030 by the transconjugants was confirmed by DNA-DNA hybridization. Resistance of transconjugants to homologous phage was complete. Curing or deletion of pTR2030 in the transconjugants confirmed that phage resistance was due to pTR2030 acquisition and not to coincident background mutation. Phage-sensitive pTR2030 deletion derivatives of LMA12 transconjugants were isolated in vivo. The HindIII fragment B of pTR2030 was subcloned into pBR322 to yield a recombinant plasmid, pMET2, useful as a source of pTR2030 DNA. A specific, chemically synthesized oligomer useful as a pTR2030 probe was derived from the sequence of a small portion of pTR2030. The conjugal strategy presented here was effective in yielding fast acid-producing, phage-resistant S. cremoris and S. lactis strains without the use of antibiotic resistance markers and without interfering with the acid-producing ability of the recipient strain.     

Transduction of a Plasmid Carrying the Cohesive End Region from Lactococcus lactis Bacteriophage ΦLC3

Plasmids carrying the cohesive end region from temperate lactococcal bacteriophage ΦLC3 could be packaged in vivo by ΦLC3 and transduced into its host strain, Lactococcus lactis subsp. cremoris NCDO 1201. The transduction frequencies were between 10^-4 and 10^-3 transducing particles per PFU, depending on the size of the phage DNA insert. This transduction system is limited to only certain lactococcal strains. The ΦLC3 cohesive site region (cos) appears to play an important role in plasmid transduction.     

Preparation and storage of high-titer lactic streptococcus bacteriophages

Various techniques were employed for preparation of high-titer bacteriophage lysates of Streptococcus lactis, S. cremoris, and S. diacetilactis strains. Infection of a 4-h host culture in litmus milk at 30 C yielded the highest titers (2 × 10⁹ to 4 × 10¹¹ plaque-forming units/ml) for most phages. Host infection in lactose-containing broth produced similar virus numbers only when 0.1 M tris(hydroxymethyl)aminomethane buffer stabilized the pH. The pH at the time of infection as well as the inoculum phage titer were critical in obtaining high titers. Optimum conditions for infection in broth were coupled with a polyethylene glycol concentration procedure to routinely produce milligram quantities of phage from 1 liter of lysate. Neutralization of whey lysates, as a means of storage, offered no survival advantage over unneutralized samples. Storage of phage lysates in a 15% glycerol whey solution at -22 C yielded a high rate of survival in most cases, even with repeated freezing and thawing, over a period of 24 months.     

Host genetic requirements for DNA release of lactococcal phage TP901‐1

The first step in phage infection is the recognition of, and adsorption to, a receptor located on the host cell surface. This reversible host adsorption step is commonly followed by an irreversible event, which involves phage DNA delivery or release into the bacterial cytoplasm. The molecular components that trigger this latter event are unknown for most phages of Gram‐positive bacteria. In the current study, we present a comparative genome analysis of three mutants of Lactococcus cremoris 3107, which are resistant to the P335 group phage TP901‐1 due to mutations that affect TP901‐1 DNA release. Through genetic complementation and phage infection assays, a predicted lactococcal three‐component glycosylation system (TGS) was shown to be required for TP901‐1 infection. Major cell wall saccharidic components were analysed, but no differences were found. However, heterologous gene expression experiments indicate that this TGS is involved in the glucosylation of a cell envelope‐associated component that triggers TP901‐1 DNA release. To date, a saccharide modification has not been implicated in the DNA delivery process of a Gram‐positive infecting phage.

We present a comparative genome analysis of three mutants of Lactococcus cremoris 3107 which are resistant to phage TP901‐1 due to mutations that affect its DNA release. Through genetic complementation and phage infection assays, we identified a novel lactococcal three‐component glycosylation system required for TP901‐1 infection. We provide new insights into the mostly unknown DNA release stage of a Gram‐positive phage, since glycosylation has not been implicated in such a process to date.     

Development of High-Frequency Delivery System for Transposon Tn919 in Lactic Streptococci: Random Insertion in Streptococcus lactis subsp. diacetylactis 18-16

The conjugative transposon Tn919, originally isolated in Streptococcus sanguis FC1, is capable of low-frequency transfer (10⁻⁷ and 10⁻⁸ per recipient) on membrane filters to a wide number of streptococcal recipients including the industrially important lactic streptococci. The introduction of pMG600 (Lac⁺ Lax⁻; a lactose plasmid capable of conjugative transfer at high frequencies and which, in certain hosts, confers an unusual clumping phenotype) into a Streptococcus lactis CH919 donor, generating S. lactis CH001, resulted in a significant improvement in the transfer frequency of Tn919 to S. lactis CK50 (1.25 × 10⁻⁴ per recipient). In addition, these matings could be performed on agar surfaces, allowing the recovery of a greater number of recipients than with filter matings. Tn919 also transferred at high frequency to S. lactis subsp. diacetylactis 18-16S but not to Streptococcus cremoris strains. Insertion in 18-16S transconjugants generated from filter matings with an S. lactis CH919 donor was random, occurring at different sites on the chromosome and also in plasmid DNA. Thus, the conditions necessary for the practical exploitation of Tn919 in the targeting and cloning of genes from a member of the lactic streptococci, namely, high-frequency delivery and random insertion in host DNA, were achieved.     

Conjugal transfer of lactose-fermenting ability fromStreptococcus lactis C2 toLeuconostoc cremoris CAF7 yieldsLeuconostoc that ferment lactose and produce diacetyl

Summary Conjugation between lactose-fermenting (Lac⁺)Streptococcus lactis C2 and Lac^– Leuconostoc cremoris CAF7 was performed. The frequency of Lac⁺ transfer was 1.5 · 10^–2 per donor cell. Lac⁺ Leuconostoc transconjugants could ferment lactose significantly faster than wild-type cells. When grown in litmus milk fortified with 0.2% yeast extract, Lac⁺ transconjugants reached pH 4.68 within 24 h at 30°C and produced diacetyl. The identity of the transconjugants asLeuconostoc derivatives was confirmed by their resistance to phage c2 and to vancomycin (>500 g/ml), and by growth on selective medium containing azide. Plasmid profiles of 10 transconjugants showed two unique patterns. A novel enlarged plasmid was found. Southern blot hybridization revealed some homology with the 30 Md Lac⁺ plasmid of donor, recipient and the transconjugants, as well as with some of the remaining plasmids of the donor.Technical Paper No. 7953, Oregon Agricultural Experiment Station.     

Phage Resistance in a Phage-Insensitive Strain of Streptococcus lactis: Temperature-Dependent Phage Development and Host-Controlled Phage Replication

Streptococcus lactis ME2 is a dairy starter strain that is insensitive to a variety of phage, including 18. The efficiency of plating of 18 on ME2 and N1 could be increased from <1 × 10⁻⁹ to 5.0 × 10⁻² and from 7.6 × 10⁻⁷ to 2.1 × 10⁻², respectively, when the host strains were subcultured at 40°C before plating the phage and the phage assay plates were incubated at 40°C. Host-dependent replication was demonstrated in N1 at 30°C and in N1 and ME2 at 40°C, suggesting the operation of a temperature-sensitive restriction and modification system in ME2 and N1. The increased sensitivity of ME2 and N1 to 18 at 40°C was also demonstrated by lysis of broth cultures and increased plaque size. ME2 grown at 40°C showed an increased ability to adsorb 18, indicating a second target for temperature-dependent phage sensitivity in ME2. Challenge of N1 with a 18 preparation that had been previously modified for growth on N1 indicated that at 40°C phage development was characterized by a shorter latent period and larger burst size than at 30°C. The evidence presented suggests that the high degree of phage insensitivity expressed by ME2 consists of a variety of temperature-sensitive mechanisms, including (i) the prevention of phage adsorption, (ii) host-controlled restriction of phage, and (iii) suppression of phage development. At 30°C these factors appear to act cooperatively to prevent the successful emergence of lytic phage active against S. lactis ME2.     

Construction of Streptococcus lactis subsp. lactis Strains with a Single Plasmid Associated with Mucoid Phenotype

Lactose-fermenting mucoid (Lac⁺ Muc⁺) variants of plasmid-free Streptococcus lactis subsp. lactis MG1614 were obtained by protoplast transformation with total plasmid DNA from Muc⁺S. lactis subsp. cremoris ARH87. By using plasmid DNA from these variants for further transformations followed by novobiocininduced plasmid curing, Lac⁻ Muc⁺ MG1614 strains containing only a single 30-megadalton plasmid could be constructed. This plasmid, designated pVS5, appeared to be associated with the Muc⁺ phenotype.