An amazing journey.

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Review in Advance first posted online on February 4, 2015. (Changes may still occur before final publication online and in print.)

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An Amazing Journey Larry McKay Annu. Rev. Food Sci. Technol. 2015.6. Downloaded from www.annualreviews.org Access provided by University of Michigan - Ann Arbor on 02/11/15. For personal use only.

Department of Food Science and Nutrition, University of Minnesota, St. Paul, Minnesota 55108; email: [email protected]

Annu. Rev. Food Sci. Technol. 2015. 6:10.1–10.17

Keywords

The Annual Review of Food Science and Technology is online at food.annualreviews.org

lactococci, plasmids, milk coagulation, aroma production, transduction, conjugation, genetics, lactose utilization

This article’s doi: 10.1146/annurev-food-022814-015713 c 2015 by Annual Reviews. Copyright All rights reserved

Abstract This article describes my early life and the chance events leading to my becoming a microbiologist and then my embarking on a career developing the plasmid biology and genetics of lactococci used in milk fermentations.

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MY LIFE: THE FIRST 18 YEARS


Why and how did I become a scientist? I attempt to present my background and the series of events that led to a successful career in the area of dairy starter cultures. The latter seemed to be by chance and serendipity. I was born in Oregon in 1943. My Dad never completed the sixth grade, and both my parents had a strong work ethic. By the time I was 10, I could saw wood with a crosscut and handle an ax to split the wood. I also worked picking berries on a farm and could “out pick” everyone except for two adult workers, and I was the only one allowed to pick in special sections with them. In essence, at an early age I, too, acquired a strong work ethic and was motivated to do my best at whatever task I encountered. When I was 11, my parents lost everything. I had come home from school to find a trailer hitched to our car with our household belongings loaded onto it. We moved around and I attended school for a week in one place and a month in another until we moved to Montana. There, my parents purchased a one-room cabin without running water, which was located on a lakefront property approximately 12 miles east of Kalispell. Our family of five lived in that one room for a year before my parents bought a home on an adjacent lot. Because of the move to Montana, I never completed the sixth grade but was allowed to start the seventh grade. The two-room school had four grades in each room and one teacher for each room. I had a stuttering problem and did not want the other kids in the school to know. So, for the first six weeks I would not respond when called on in class. When asked about this at the first parent/teacher conference, my mom answered that I was probably afraid of stuttering. The teacher never pushed the issue, and eventually I was comfortable responding in class. Even then I must have been a determined individual! Although I never stuttered again, this early issue is likely related to my feeling comfortable advising students (see Table 1) one-on-one but not always feeling comfortable speaking to groups of people, unless I am well prepared. For the next six years, living by the lake and mountains was paradise, a dream (swimming, ice skating, fishing, water and snow skiing, hiking in the mountains, and seeing the local wildlife). The home my parents had purchased was a small two-bedroom dwelling, so my brother and I slept in the attic above the garage. We slept there year-round, even though the space was unheated. The Montana winters do indeed get cold, and the coldest night in the five years we slept there was minus 39◦ C. It must have been good for my health because from the eighth grade to the end of high school I never missed a day of school. After I was in my profession, my mom told me that upon graduating from the eighth grade, the high school counselor called my parents repeatedly, insisting I needed to pursue a different route than the science and math curriculum I had signed up for, because I would not be able to succeed in that area. The calls came to an end when he called my dad at work and was told we did not want to hear from him again. I went from a class size of seven in the eighth grade to a freshman class of more than 300 in the county high school. I graduated in the top 10% of my class in 1961. The final exam for my junior-year trigonometry course was 50 problems solved in class during each of the last five classes of the quarter. As I walked into class on the last day of the exam, the teacher grabbed me by the back of the neck and in front of the class commenced to tell the students that in all the years he had taught the class, I was the only one to come into the exam on the last day and have 45 out of the possible 45 correct answers up to that point. I was so embarrassed, even quivering, when I reached my desk that I missed three of the final five problems to be solved that day! In high school I was sports editor of our class yearbook and was selected as a delegate to attend Montana Boys State. I had not considered attending college until one of my friends asked me if I would be his roommate at the University of Montana. He convinced me to go to college. At the exit interview with the high school counselor, I was told I should attend a trade school and that college would be very difficult for me. He could not have been more wrong!

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Table 1

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List of advisees and their accomplishments

MS theses

PhD theses


A. Chudgar, 1972 (Plan B)

B.R. Cords, 1973a

R.F. Schifsky, 1973

J. O’Leary, 1974 (co-advisor)

C. Park, 1974

R.H. Schmidt, 1974 (co-advisor)

J.D. Efstathiou, 1974b

J.D. Efstathiou, 1977

B.E. Schmidt, 1975 (co-advisor)

T.R. Klaenhammer, 1978

T.R. Klaenhammer, 1975

L.D. Larsen, 1979

D.G. Anderson, 1976 (microbiology)

G.M. Kempler, 1980c

G.L. Mays, 1978

P.M. Walsh, 1981 (genetics)

P.T. Zoltai, 1980 (Plan B; co-advisor)

D.G. Anderson, 1983 (microbiology)f

R. Snook, 1981

S.K. Harlander, 1984d

T.W. Wolfe, 1983

J.K. Kondo, 1984

D. Romero, 1984

K.S. Harmon, 1986e

J.L. Steele, 1985

J.L. Steele, 1989 (genetics)

K. Polzin, 1985 (microbiology)

J.M. Feirtag, 1990

M.J. Bohanon, 1986 (Plan B)

B.R. Froseth, 1990h

B.R. Froseth, 1986

K.M. Polzin, 1991 (microbiology)

J.M. Feirtag, 1986g

G. Stoddard, 1991

K.M. Kolaetis, 1989

L.A. McLandsborough, 1993

B.F. Hughes, 1991

W. Yu, 1994

T.L. Britton, 1992

H.R. Riepe, 1996

H.R. Riepe, 1992

D.A. Mills, 1995 (microbiology; co-advisor)i

E. Pasalodos, 1993

H. Wang, 1998j

C. Sanchez, 1994

P. Ray, 2000

K. Wahls, 1994

T. Phister, 2001

M. Polzin, 1998 a

In 1973, B. Cords received an American Society for Microbiology President Fellowship to spend two weeks at the University of Washington to acquire skills in plasmid DNA research. b In 1976, J.D. Efstathiou placed second in a graduate student scientific paper presentation contest at the American Dairy Science Association (ADSA) meeting. c In 1980, G.M. Kempler received the Richard M. Hoyt Memorial Award from the ADSA for her outstanding graduate research. d In 1982, S.K. Harlander placed second in a graduate student scientific paper presentation contest at the ADSA annual meeting. e In 1983, K.S. Harmon placed second in a graduate student scientific paper presentation contest at the ADSA annual meeting. f In 1984, D.G. Anderson received the Bacaner Award in Microbiology (outstanding graduate student award) from the Department of Microbiology. g In 1986, J.M. Feirtag placed first in a graduate student scientific paper presentation contest at the ADSA annual meeting. h In 1988, B.R. Froseth placed first in the Z. John Ordal microbiology oral paper competition at the Institute of Food Technologists’ annual meeting. i In 1995, D.A. Mills received the University of Minnesota Sigma Xi Graduate Student Research Award and the Bacaner Research Award from the Department of Microbiology for being an outstanding graduate student; in 1996, he received the Graduate Student Research Award at the American Society for Microbiology’s North Central Branch’s annual meeting. j In 1997, H. Wang placed third in a graduate student scientific paper presentation contest at the ADSA annual meeting.

www.annualreviews.org • An Amazing Journey


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MY UNDERGRADUATE COLLEGE YEARS


I started at the University of Montana majoring in wildlife technology and at some point switched to forestry. During the summers at the end of my freshman and sophomore years, I worked for the Montana State Forestry Department where I had lookout duties, fought fires, cleared trails, etc. In my freshman year, the dormitory resident assistant was majoring in microbiology. He kept telling me about the subject, so during my sophomore year, I took a microbiology course. I was hooked! It was fascinating, and I kept taking more microbiology courses and ultimately graduated in 1965 with honors in microbiology with a minor in chemistry. Perhaps microbiology intrigued me given its immediate application to everyday life or given that as a child, I had mumps, measles, whooping cough, and chicken pox as well as a case of food poisoning. The latter was still vivid in my mind, because approximately two weeks prior to the end of the fourth grade, this single case of food poisoning caused me to lose my perfect-attendance status at school. In the microbiology courses, I not only learned about these diseases but also realized I had probably had staphylococcal food poisoning based on what I had eaten and the symptoms that followed. In my third year at the university, I served as a resident assistant in the dormitory. During my junior year, the instructor in one of my microbiology classes asked to see me in his office. I did not know what to expect but he wanted to know if I would like to be a National Science Foundation Undergraduate Research Participant during the summer and into my senior year. It only paid $20 a month but I spent up to 40 hours a week on a research project during that summer. Because I was going to be married to my then fiancée Dixie that September (we celebrated our 50th anniversary in 2014), I also needed another job, so I worked weekends at a saw mill. Initially, I had no intention of attending graduate school but while working in the lab that summer I realized I was just as good as, or better than, some of the graduate students pursuing their degrees. Thus, I applied to graduate school and accepted a research assistantship in the area of dairy starter cultures from the Department of Microbiology at Oregon State University, under the direction of Dr. William Sandine. I was schooled in medical microbiology but leaned toward industrial microbiology as a possible career.

GRADUATE AND POSTGRADUATE TRAINING I started as an MS student in July of 1965. At a research meeting with Dr. Sandine in early 1966, he asked whether I would be interested in going directly into a PhD program. I switched. I couldn’t stay away from the lab—it was that interesting. I presented a scientific paper at the Annual Meeting of the American Dairy Science Association (ADSA) within a year of entering graduate school. At the end of my first microbiology class (required for all incoming graduate students), I went to the Professor to get my grade. He laughed and told me I could have taught the class. I always liked to study, and that, too, showed how excellent my training in microbiology had been at the University of Montana. My PhD dissertation was on the biochemical and genetic mechanisms of lactose utilization by lactococci. We found that the assumed labile nature of β-galactosidase in these organisms was due to the presence of a phosphoenol pyruvate-dependent phosphotransferase system. Instead of hydrolyzing lactose, the lactococci possessed a phospho-β-galactosidase that hydrolyzed lactosephosphate, but not lactose (McKay et al. 1969, 1970). Our initial progress on this problem was clarified when Hengstenberg et al. (1967) reported that Staphylococcus aureus utilized lactose via a phosphoenol pyruvate-dependent phosphotransferase system and instead of hydrolyzing lactose, they possessed a new enzyme hydrolyzing the phosphorylated derivative of lactose. I was in the right place at the right time. Although the significance was not realized at the time, we also 10.4

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observed that if lactococci were grown on glucose as a carbohydrate source, they lost the ability to ferment lactose and simultaneously lost the three specific enzymes required for lactose utilization. After completing my PhD in 1969, I went to the Department of Microbiology at Michigan State University as a postdoctoral student to work with Dr. Ralph Costilow on Bacillus popilliae, an insect pathogen. A PhD student there originally from England insisted that I read a paper by Hirsch (1952) on the evolution of lactic streptococci (now termed lactococci). I finally stopped by the library one day on my way home from work and ended up spending the next four hours going from one library to the next looking up publications Hirsch had cited. Hirsch’s publication set the stage for my career in the field of lactococcal genetics. Hirsch proposed that lactococci were evolving to milk as their new ecological niche, based largely on their instability of lactose utilization. He then cited publications from the 1930s describing that when lactococci were grown on any carbohydrate other than lactose or galactose, they lost their ability to ferment lactose. It had also been known since the 1930s that the proteolytic activity of lactococci could be spontaneously lost, resulting in slow acid production. Certain strains of lactococci could also spontaneously lose their ability to ferment citrate and hence to produce butter aroma. From the above observations and recalling some results from my PhD research, I had an aha! moment when I realized the instability of lactose utilization, proteolytic activity, and citrate fermentation were probably linked to the loss of plasmid DNA elements. Plasmids are small circular pieces of DNA that exist in the cell separate from the chromosome and were at the time becoming an active area of research in other bacteria, especially as related to the acquisition of antibiotic resistance. When I finally returned home four hours late and told my wife what I had read and what it might mean, she was not impressed. I was late! After a year as a postdoctoral student, I was fortunate to be offered a position as an assistant professor in the Department of Food Science and Nutrition at the University of Minnesota. They were searching for someone to conduct research in the area of dairy starter cultures. How fortuitous!

MY CAREER AT THE UNIVERSITY OF MINNESOTA Setting the Stage My career at the University of Minnesota started in August of 1970. I wanted to develop a program on the physiology and genetics of lactococci to possibly improve their functionality in milk fermentations. A key component was the examination of these organisms for the presence of plasmid DNA and its relationship to instability of functional properties essential for successful milk fermentations. Genetic and molecular biology studies on microorganisms important in food microbiology were still sometimes considered irrelevant. I was told by a leading individual in the field of dairy starter cultures when I began this research that perhaps another area of food microbiology would be more beneficial for publishing and obtaining tenure. He indicated that dairy starter cultures had already been thoroughly investigated. I did not respond because I believed it was a field ready for the application of genetic tools that could be used to improve and stabilize the industrial performance of starter cultures. From 1970 to 1978, we were the only laboratory working in this area, worldwide. During these initial years, our research findings were described by some as chance, fortuitous, and serendipitous.

Setting the Stage for Plasmid Studies We began our studies by confirming that strains of Lactococcus lactis subspecies L. lactis, L. cremoris, and L. diacetylactis could spontaneously lose their ability to ferment lactose and that plasmid curing www.annualreviews.org • An Amazing Journey


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agents enhanced this loss (McKay et al. 1972). This provided phenotypic evidence for plasmidmediated lactose utilization. Bruce Cords, a PhD student, went to Dr. Stanley Falkow’s laboratory at the University of Washington to learn plasmid isolation techniques, and I followed later to learn electron microscopic techniques necessary to observe individual plasmids. At the time, plasmid DNA was isolated by labeling the DNA with radioactive thymidine, extracting the DNA from the cells, separating plasmid DNA from chromosomal DNA by cesium chloride-ethidium bromide density gradient centrifugation, and then examining for plasmids with electron microscopy—a procedure requiring two to three weeks to determine the plasmid profile of a strain. Lactococcal strains were shown to contain a diversity of plasmid sizes (Cords et al. 1974, McKay & Baldwin 1975, Anderson & McKay 1977, Larsen & McKay 1978, Kempler & McKay 1979a). Some of these studies were facilitated when Todd Klaenhammer, a PhD student, developed an improved method for isolating plasmid DNA from lactococci (Klaenhammer et al. 1978). The development of this lysis method was critical because of the unique cell wall properties of lactococci that were not amenable to the few plasmid isolation methods available at the time. This procedure, coupled with a method for separating plasmid DNA based on size using agarose gel electrophoresis (Guerry et al. 1973), reduced the time for identifying plasmids from weeks to a day and paved the way for rapid plasmid analysis in lactococci. Also, many of the plasmids harbored by lactococci were large and difficult to isolate, and thus required the development of special techniques. This was accomplished by Anderson & McKay (1983). It was subsequently found that lactose-negative (Lac− ) derivatives of L. lactis C2 and other lactococcal strains were missing a single plasmid (McKay & Baldwin 1974b, Anderson & McKay 1977, Kempler & McKay 1979b, Kuhl et al. 1979). This provided physical evidence for the linkage of lactose utilization to plasmid DNA.


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Lysogeny Concomitant with the above studies, we initiated a study on lysogeny in lactococcal strains. Lysogeny refers to the phage carrying state of many bacterial strains wherein a complete copy of the phage genome (termed prophage) is silently carried in and replicated along with the bacterial chromosome. There were limited reports on the occurrence of lysogenic lactococcal strains, which spontaneously released their phage, causing cell lysis. We found using UV irradiation as an inducing agent increased the chances of finding lysogeny in lactococci. In fact, all strains examined were found to be lysogenic. As a negative control we attempted to use L. lactis C2, a strain commonly used in research, but it was also lysogenic and released phage upon lysis, as observed by electron microscopy (McKay & Baldwin 1973). However, no indicator strain for the phage was found until 1987 when Baldwin (see Figure 1) made a chance observation (Baldwin & McKay 1987). It was also confirmed, using prophage-inducing agents, that some commercial dairy starter cultures produced in the United States contained phage-harboring strains (Park & McKay 1975).

DEVELOPMENT OF GENE TRANSFER SYSTEMS IN LACTOCOCCI Transduction Transduction is a gene transfer system in which phage released from a lysogenic strain is used to transfer genetic material from one cell to another and had not yet been demonstrated in lactococci using temperate phages. The dairy industry had done little to improve the strains they depended on, perhaps due to the absence of a genetic system for such studies. By using the phage lysate induced from the lysogenic L. lactis C2, the transduction of lactose utilization was 10.6

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Figure 1 Kathy Baldwin and Larry McKay in the lab.

demonstrated, thus establishing a gene transfer system that could be used to examine the genetics and the instability of lactose utilization in this strain (McKay et al. 1973, McKay & Baldwin 1974b). This discovery was by chance, given that it was the first and for a long time only strain we examined for transducing ability. A plasmid-free and prophage-cured derivative of L. lactis C2 was then isolated after two years of attempts and examining thousands of isolates. This strain was instrumental in our future genetic studies and was initially used to confirm by transduction that lactose utilization was genetically linked to plasmid DNA (McKay et al. 1976). Later, in 1981 we reported that L. cremoris C3 was lysogenic and the induced phage could also transfer a Lac plasmid to L. lactis, demonstrating inter- and intraspecies genetic transfer in dairy starter cultures (Snook & McKay 1981).

Conjugation Conjugation is the transfer of genetic material from one cell to another as a result of cell-to-cell contact. We first reported possible conjugation of lactose-fermenting ability in 1979 (Kempler & McKay 1979b). I was in the laboratory and asked Gail Kempler about her attempted conjugation trials. She said it did not work and that the control plates were negative but one lactose-fermenting colony appeared on the conjugation plate. She said she discarded the plates. I am not sure what I said but when I left the lab, Kathy Baldwin told her to retrieve the plate and examine the colony for the possible transfer of the lactose plasmid. Conjugation in lactococci was discovered! We then showed that conjugation was common among lactococcal strains (McKay et al. 1980, Snook & McKay 1981). In 1980, workers in England also exhibited conjugation in L. lactis (Gasson & Davies 1980). They found high-frequency conjugation associated with cell aggregation. We then found that some strains possessed a transfer factor (a plasmid) that combined with the lactose plasmid, resulting in cell aggregation and high-frequency transfer of lactose utilization (Walsh & McKay 1981). This work also provided the first indirect evidence for the presence of transposable www.annualreviews.org • An Amazing Journey


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elements in lactococci and directly led to the isolation and sequencing of the first insertion sequence (IS) in lactococci by Polzin and Shimizu-Kadota in Japan (Anderson & McKay 1984, Polzin & Shimizu-Kadota 1987). A key practical application of conjugation was that strain construction could proceed by natural conjugal transfer of key phenotypes into select strains (Steele & McKay 1989), a common practice in today’s starter culture strain development programs. Additional work examined the transfer factor, termed pRS01, in which the regions responsible for high-frequency conjugation and a cell aggregation phenotype (Clu+ ) were mapped (Anderson & McKay 1984). The aggregation phenotype of the L. lactis transconjugants was unstable and was shown to undergo a phase variation between Clu+ and Clu− . This phase variation was due to the inversion of a DNA sequence in the aggregation gene on the plasmid, the first such invertible element described in lactococci. Because the Clu− strains transferred their plasmids at a low frequency, the inversion element also influenced transfer ability. Further mapping of pRS01 identified additional regions involved in the transfer phenotype (Mills et al. 1998), including the conjugative origin of transfer as well as a gene involved in preparing the plasmid for conjugal transfer. The origin of the transfer region was also shown to contain a group II intron, the first functional group II intron to be characterized in eubacteria (Mills et al. 1996, 1997). Group II introns are mobile retroelements that, in addition to splicing, home into intronless alleles via an RNA intermediate. By providing a bacterial context in which to study group II intron function, the discovery of the lactococcal intron ushered in an explosion of fundamental studies on group II intron biology (Lambowitz & Zimmerly 2004). Moreover, the lactococcal intron originally identified in pRS01 is now sold commercially by Sigma Chemical Company as a means of generating targeted insertions within numerous bacterial genera.


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Protoplast Transformation The third mechanism of genetic transfer developed in our laboratory was protoplast transformation (Kondo & McKay 1982, 1984). At the time, the development of protoplast transformation provided a way to introduce both natural and engineered DNA into L. lactis. This was a major contribution for the engineering of lactococcal starter cultures and opened the door to cloning studies in these bacteria. The development of this technique required the discovery of several new methods for handling lactococci, including generating protoplasts, handling the fragile protoplasts in such a way that they did not lyse, inducing expression of marker genes, and regeneration of protoplasts into viable cell lines. Electroporation techniques to introduce DNA into lactococci were introduced and eventually became the preferred and more generic method for transformation across Gram-positive bacteria (McIntyre & Harlander 1989).

APPLYING PLASMID BIOLOGY, MICROBIAL PHYSIOLOGY PRINCIPLES, AND GENETICS TO LACTOCOCCI Stabilization of Lactose Utilization With the knowledge that lactose-fermenting ability is plasmid mediated and with the discovery of a transduction system, a lactose-fermenting transductant was isolated from L. lactis C2 in which the lactose utilization genes were stabilized (McKay & Baldwin 1978). It was shown that the lactose plasmid or a portion of it had become integrated into the chromosome of the transductant. Thus, unstable plasmid-linked traits like lactose utilization could be stabilized by a natural gene transfer mechanism like transduction and provide the conceptual basis for stabilizing other traits that are plasmid mediated and vital for successful milk fermentations. 10.8

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Aroma Production The use of L. diacetylactis as a flavor producer in dairy fermentations depends on its ability to produce diacetyl from citrate. However, it was well known from the early literature that these strains can spontaneously lose their ability to produce butter aroma due to loss of citrate utilization. This spontaneous loss had not been explained. We correlated the loss of citrate utilization and, thus, loss of ability to produce butter aroma to the loss of plasmid DNA in these strains (Kempler & McKay 1979a). These genetic studies were facilitated by the development of a modified medium that distinguished citrate-fermenting (blue colonies) from noncitrate-fermenting variants (white colonies) within 48 h (Kempler & McKay 1980). In addition, this medium served as a quick means for distinguishing L. diacetylactis from other lactococci in multiple-strain starter cultures. It had been reported that certain L. lactis and L. cremoris strains can produce aroma compounds and excessive amounts of carbon dioxide (Krishnaswamy & Babel 1951, Sandine et al. 1957). The reason for the spontaneous appearance of these altered strains was not known. With her keen sense of observation, Kathy Baldwin noted and isolated an abnormally large colony of L. lactis C2. When she first showed me the colony, I told her it was a contaminant because lactococci do not form such large colonies. She never said anything but after a few weeks brought back data convincing me it was a spontaneous variant of C2. It was subsequently shown to produce excessive carbon dioxide, more than 1,000 μg/ml of Westerfeld-positive material (sum of acetoin plus diacetyl) and exhibited slow lactic acid production due to a defect in the enzyme lactic dehydrogenase (McKay & Baldwin 1974a). It indicated a type of mutation that could be responsible for the spontaneous appearance of aroma- and carbon dioxide–producing variants of L. lactis or L. cremoris.

Loss of Fast Milk Coagulating Ability Proteolytic activity is required for rapid acid production when lactococci are grown in milk, given that they depend on this activity to obtain needed nitrogenous compounds in the form of peptides and amino acids from caseins, which are the predominant proteins in milk. Proteolytic-deficient variants of lactococci that exhibit a slow milk coagulating ability were known since the 1930s to occur spontaneously. We noted that the genetic locus for proteinase activity was linked to lactose utilization when both phenotypes could be cotransduced into other strains (McKay & Baldwin 1974b). Later, we determined that the genes responsible for proteinase could be on the same plasmid as the genes responsible for lactose utilization (Kuhl et al. 1979, McKay & Baldwin 1975). Stoddard & Richardson (1986) observed that some assumed proteinase-deficient variants of lactococci actually possessed proteolytic activity. They suggested these strains were unable to transport or utilize the released nitrogenous compounds from casein for growth. In 1993, workers in the Netherlands sequenced a chromosomal fragment from L. lactis that coded for an oligopeptide permease system that was required, in addition to the proteinase, for the utilization of casein as a nitrogen source and for fast milk coagulation (Tynkkynen et al. 1993). In collaboration with workers at Utah State University, we established that the oligopeptide permease system was also linked to plasmids in many lactococci (Yu et al. 1996). We also described another reason for the slow milk coagulating phenotype in L. lactis (Wang et al. 1998). This variant exhibited slow milk coagulation even though it possessed all the known components essential for utilizing casein as a nitrogen source, which included a functional proteinase and oligo-, di/tripeptide, and amino acid transport systems. The variant, by determining its amino acid requirements, was shown to require aspartic acid to exhibit fast milk coagulation. The variant was shown to be missing pyruvate carboxylase and, hence, was unable to form oxaloacetate and, hence, aspartate from pyruvate and carbon dioxide. This result suggested that when www.annualreviews.org • An Amazing Journey


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lactococci are grown in milk, aspartic acid derived from casein is unable to fully meet the nutritional demands of lactococci, and thus they become dependent on aspartate biosynthesis.

Bacteriocin Production


Many lactococci are known to produce proteinaceous antimicrobial substances referred to as bacteriocins. Using conjugation, we showed that a bacteriocin produced by an L. diacetylactis strain, active against other lactococci, was linked to a large plasmid (Scherwitz et al. 1983). We then attempted to clone the bacteriocin-producing gene cluster by using protoplast transformation, but after a year with no results the student was ready to give up. I told her to finish examining the remaining transformants from the last trial and if one was not found we would move on. A bacteriocin-producing transformant was found (Scherwitz-Harmon & McKay 1987). In collaboration with workers from the Netherlands, this bacteriocin gene cluster was sequenced, characterized, and determined to be lactococcin (Stoddard et al. 1992). These studies showed the benefits of using conjugation and transformation to examine the genetics of lactococci and that more stable starter blends could be achieved by eliminating bacteriocin-producing plasmids from desired strains.

ISOLATION OF POTENTIAL STRAINS FOR ENHANCED CHEESE FLAVOR The development of methods to reduce the ripening time for flavor development of Cheddar cheese has been a coveted goal of the cheese industry. We approached the problem by isolating an L. lactis variant that would lyse (thermolytic) at the cooking temperature (38◦ –40◦ C) used in cheese manufacture (Feirtag & McKay 1987a). Such variants could possibly accelerate cheese ripening either by releasing their intracellular enzymes into the curd at an early stage in the cheesemaking process or by serving as a delivery system of ripening enzymes, the genes of which could be introduced into the variant. To illustrate the feasibility of using this system, we introduced a plasmid containing the cloned neutral protease gene from Bacillus subtilis that had been constructed by workers in the Netherlands (van de Guchte et al. 1990) into the thermolytic variant. A transformant was isolated that actually overproduced the neutral protease and clarified milk in five days at 21◦ C, indicating the variant could potentially be used as a delivery system (Riepe & McKay 1994). It was also found that temperature-sensitive strains of L. cremoris were naturally thermolytic at the cooking temperature (Feirtag & McKay 1987b). Autolysis of lactococcal strains used as starters in cheese manufacture may play a key role in cheese ripening by also releasing the cells’ intracellular enzymes into the cheese matrix (Crow et al. 1995). In collaboration with workers at the New Zealand Dairy Research Institute and as part of my sabbatical leave there, lactococcal strains were screened for autolysis and two were found to exhibit high activity. This autolytic system was then characterized (Riepe et al. 1997). Subsequently, using protoplast transformation we found that the lactose plasmid from temperature-sensitive L. cremoris SK11 conferred a temperature-sensitive phenotype to L. lactis (Feirtag et al. 1991). This finding could increase the number of strains available for cheese manufacture and could reduce the tendency for bitterness in cheese when L. lactis strains are used for cheesemaking. This is because strains that grow at the cooking temperature (38◦ –40◦ C) used in manufacture of Cheddar cheese leads to high total proteinase activity, which in turn can contribute to the development of bitterness. Thus, strains whose growth is repressed at the cooking temperature, such as temperature-sensitive L. cremoris, are usually preferred in Cheddar cheese manufacture. 10.10

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DEVELOPMENT OF FOOD-GRADE CLONING AND INTEGRATION VECTORS Following the development of the protoplast transformation system, we constructed the first foodgrade cloning and integration vectors for gene stabilization in lactococci. This became feasible after we isolated a plasmid from L. diacetylactis coding for nisin resistance (McKay & Baldwin 1984). This nisin-resistant determinant was cloned onto a small DNA fragment that was also found to contain an origin of replication that allowed the fragment to exist as an independent replicon (Froseth et al. 1988b). On the basis of these findings, a first-generation cloning vector applicable to the food processing industry was constructed (Froseth & McKay 1991). A second potential food-grade marker that was cloned in our laboratory was the β-galactosidase gene from the chromosome of Streptococcus thermophiles (Herman & McKay 1986). The construction of cloning vectors that could integrate into the lactococcal chromosome for use in commercial cultures would allow stabilization of important metabolic properties. These integration vectors were being developed in several laboratories (Chopin et al. 1989, Leenhouts et al. 1990). We had cloned and localized the origin of replication with its temperature-sensitive maintenance regions from the lactose plasmid in L. cremoris SK11 (Feirtag et al. 1991, Horng et al. 1991) and had also reported the isolation of a lactococcal IS element, which was present in multiple copies in the chromosome of many lactococci, including those used in commercial strains (Polzin & McKay 1991). We then combined a fragment of this IS element with the naturally occurring temperature-sensitive maintenance region of replication to produce the first lactococcal integration vector based on recombination with chromosomal IS elements (Polzin & McKay 1992).

PLASMIDS CODING FOR BACTERIOPHAGE RESISTANCE The conjugative plasmid coding for nisin resistance mentioned above was also able to confer insensitivity to phage infection at 21◦ C and 32◦ C but not at 37◦ C (McKay & Baldwin 1984). We suggested that the finding of a phage-insensitive determinant located on a conjugative plasmid should prove useful in constructing phage-insensitive strains for dairy fermentation processes. Although we later found other plasmids coding for phage insensitivity (Laible et al. 1987; Froseth et al. 1988a; Murphy et al. 1988; Steele et al. 1989; McKay et al. 1989; McLandsborough et al. 1995, 1998), this area, showing that plasmids in lactococci coded for a variety of phage-insensitive mechanisms and the construction of phage-insensitive strains based on acquisition of plasmids coding for phage insensitivity, was developed and pioneered by Todd Klaenhammer’s group at North Carolina State University.

TEACHING AND GRADUATE EDUCATION For many years I taught an introductory microbiology course, but my primary teaching responsibilities included a required senior/graduate level course on the microbiology of food fermentations and an advanced graduate level course on microbial starter cultures, which I developed in 1975. The subject material in the latter course became one of the most exciting research areas in food microbiology worldwide, i.e., the physiology, genetics, and application of lactic acid bacteria used in food fermentations and human health. For each subject area, I presented a historical background and then proceeded to the latest information. I tried to present the logic, philosophy, and experimental approaches used by different investigators to advance the subject area. This, in turn, helped the students approach their own research. Two student comments from class evaluations www.annualreviews.org • An Amazing Journey


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sum up this course: “. . .It was my first introduction to bacterial genetic research, it was hard as hell, and I loved it,” and, “Today we would say that Dr. McKay taught critical thinking skills; back in the 1980s we simply knew that while Dr. McKay’s courses were the hardest courses we would take, they were also courses where we learned the most about being a scientist.” I was blessed with many superb graduate students, and in many cases I struggled to keep up with their progress. Some even told me to stay out of their way! What were my teaching/mentoring styles for graduate education? In truth, I don’t know, but they were probably very similar to those of other successful graduate teaching and research programs. I believe that one’s graduate students cannot be treated as a collective group, but each must be treated as an individual. Each student has her/his unique background abilities, and I tried to get each to reach her/his fullest potential. It was my job to provide the environment in which this could happen. Each student was encouraged to read, not only the current literature pertaining to their research project but also the historical literature to gain a complete grasp of the project. They then needed to acquire and apply the latest techniques and knowledge to pursue their research objectives. Coursework is important for the latter. A student’s mind must be prepared to make observations and to create linkages from different pieces of knowledge, i.e., to develop critical thinking. For MS students, I tended to provide extensive guidance, as their primary objective was to gain additional knowledge in a particular field and to learn how to conduct research. For a PhD student, the objectives were similar, but they must also develop independent thinking. After completing an MS program with me, a student was shocked when a few months into his PhD program I turned the tables on him. I wanted him to tell me the rationale, logic, and background for the experiments he was reporting on; what the data meant; and what follow-up experiments he would conduct. He was able to do this quickly and was soon advising me on the project. This was true for many of my PhD students—they became excited, motivated, and dedicated knowing it was about their careers, and they had the ability to make themselves successful. I provided opportunities for my students, continually asked for clarifications, provided constructive criticism, and constantly coached and encouraged them. The students came to see that they must believe in their own abilities. Nothing can be more fulfilling than to see the joy and excitement of a student who has succeeded. Encouragement in research is essential, whether it be to push a student to travel to learn a new research technique, to nudge the student in a different direction, to take a step back, or to convince a student to ask a little more of themselves—the point is that I believed in working closely with students but at the same time encouraging them to become independent investigators and individuals. I have been asked how my students are able to get jobs in the food industry, as the duties of such positions are not necessarily related to their thesis work. When I asked my students about this, their response was that it did not matter what they worked on because they were trained to investigate and solve problems.


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Travel Highlights of my career included being invited to present 26 international lectures in Ireland, England, Argentina, France, South Korea, the Netherlands, Canada, Mexico City, Austria, Japan, Greece, Thailand, New Zealand, Australia, and Russia. These were all fantastic experiences. For the journey to Tucumán, Argentina in 1981, I was selected by the American Society for Microbiology (ASM) International Professorships Program for Latin America to teach a three-week course on lactic starter cultures. Between 2004 and 2005, I spent a sabbatical leave at the New Zealand Dairy Research Institute, which was also a fantastic experience.

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Research and Teaching Awards I received numerous honors and awards in recognition of research and teaching contributions, including the 1976 Pfizer Award, the 1982 Dairy Research Foundation Award, the 1985 American Cultured Dairy Products Institute Research Award, the 1987 Fisher Award from the ASM, and the 1990 Borden Award from the ADSA. From 1989 to 1994, I was the Kraft General Foods Chair in Food Science from the Institute of Food Technologists (IFT) and was named a fellow of IFT in 1991. In 1992, I was the recipient of the Alexander von Humboldt Foundation Award for outstanding contributions to American agriculture. In 1991, I received the Oregon State University Alumni Fellow Award. At the University of Minnesota, I received the 1996 College of Human Ecology Excellence in Research Award, the 1991 Award of Merit from Gamma Sigma Delta, a 2001 Award for Outstanding Contributions to Graduate Education and was inducted into the University’s Academy of Distinguished Teachers. In 1994 I received a Fulbright Research Travel Award for a sabbatical leave at the New Zealand Dairy Research Institute and in 2007 the Elie Metchnikoff Prize in Biotechnology from International Dairy Federation.

CONCLUDING REMARKS Even though lactococci had been and were being extensively investigated worldwide because of their importance in milk fermentations, from 1970 to 1978 we were the only known laboratory working on the plasmid biology and genetics of these bacteria. I was lucky to start at the very beginning of this field. Our initial findings provided the foundation for a field of research that began to expand rapidly. Mike Gasson (England) entered the field around 1978 and was followed soon thereafter by Gerald Venema and Willem de Vos in the Netherlands, and Todd Klaenhammer at North Carolina State University. The field then exploded and now occupies hundreds of researchers and industry professions in laboratories all around the world. These initial studies paved the way for the once genetically obscure and essentially genetically unstudied lactococci becoming one of the best studied bacteria and model organisms in the world, and for developing an understanding of Gram-positive bacterial genetics. In addition, subsequent work by scientists around the world laid the foundation for the current genomic and metabolic analysis being done in lactococci and other lactic acid bacteria. When I started in 1970, I wanted to develop a program on the genetics of lactococci to possibly improve their functionality in milk fermentations. I felt it was an area ready for genetic application if the genetic tools could be developed. This happened and the field became one of the most exciting areas of research to pursue. As the field began to grow, the camaraderie among the individuals was great. It was like a small fraternity of scientists advancing the field together. This sense of fraternity has been perpetuated by an international symposium titled “Lactic Acid Bacteria: Genetics, Metabolism, and Applications,” which was established in the Netherlands in 1983 and has been held there approximately every three years since. After completing work with my last two PhD students and upon the retirement of Kathy Baldwin, I, myself, retired in July of 2004. It was an amazing journey!

DISCLOSURE STATEMENT The author is not aware of any affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this review.



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ACKNOWLEDGMENTS


My life over the years has been more or less dictated to growing cultures of lactococci. This could not have been accomplished without the support of my wife and two sons. The latter told me after entering their careers that I never did have a job, but rather a hobby. I extend my deepest gratitude to the graduate students I advised. The breakthroughs accomplished were due to their dedication, creativity, persistence, perseverance, scientific inquisitiveness, and commitment to their projects. I extend thanks to the postdoctoral fellows (Mary Harbough, Richard Herman, Jason Horng, Nancy Laible, Jim Petzel, Teresa Requena, L. Sechaud, and Craig Schroeder) for bringing their talents to our lab and for their help and major contributions in our research program. Thanks to Dr. Gary Dunny for his discussions on bacterial genetics and collaborative studies. Thanks go to all the visiting scientists and sabbatical leave personnel for bringing their experiences to our lab. The assistance of other staff and undergraduates that worked in the lab is also greatly appreciated. I am especially indebted to Dr. Sandine, my PhD advisor. I could not have been tutored by a more supportive, enthusiastic, or positive person. He provided the atmosphere and opportunity for individuals to excel. Finally, I would like to thank the Department of Food Science and Nutrition and the Minnesota Agricultural Experiment Station at the University of Minnesota for their support throughout my career. They made it possible to succeed. This manuscript is dedicated to Kathy Baldwin and to the graduate students I advised. Kathy Baldwin was a dedicated and talented scientist whose acute observations made possible many original research contributions. She also managed our laboratory for more than 33 years and was an asset for maintaining continuity in the laboratory. Kathy Baldwin and the graduate students were colleagues in a research adventure and they were the ones responsible for the research accomplishments. They made my career possible. LITERATURE CITED Anderson DG, McKay LL. 1977. Plasmids, loss of lactose metabolism and appearance of partial and full lactose fermenting revertants in Streptococcus cremoris B1 . J. Bacteriol. 129:367–77 Anderson DG, McKay LL. 1983. Simple and rapid method for isolating large plasmid DNA from lactic streptococci. Appl. Environ. Microbiol. 46:549–52 Anderson DG, McKay LL. 1984. Genetic and physical characterization of recombinant plasmids associated with cell aggregation and high-frequency conjugal transfer in Streptococcus lactis ML3. J. Bacteriol. 158:954– 62 Baldwin KA, McKay LL. 1987. Spontaneous release of temperate phage by relysogenized lactose-positive transductants of Lactococcus lactis C2. J. Dairy Sci. 70:2005–12 Chopin M-C, Chopin A, Rouault A, Calleron N. 1989. Insertion and amplification of foreign genes in the Lactococcus lactis subsp. lactis chromosome. Appl. Environ. Microbiol. 55:1769–74 Cords BR, McKay LL, Guerry P. 1974. Demonstration of extrachromosomal elements in group N streptococci. J. Bacteriol. 117:1149–52 Crow VL, Coolbear T, Gopal PK, Martley FG, McKay LL, Riepe HR. 1995. The role of autolysis of lactic acid bacteria in the ripening of cheese. Int. Dairy J. 5:855–75 Feirtag JM, McKay LL. 1987a. Isolation of Streptococcus lactis C2 mutants selected for temperature sensitivity and potential use in cheese manufacture. J. Dairy Sci. 70:1773–78 Feirtag JM, McKay LL. 1987b. Thermoinducible lysis of temperature sensitive Streptococcus cremoris strains. J. Dairy Sci. 70:1779–84 Feirtag JM, Petzel JP, Pasalodos E, Baldwin KB, McKay LL. 1991. Thermosensitive plasmid replication, temperature-sensitive host growth, and chromosomal plasmid integration conferred by Lactococcus lactis subsp. cremoris lactose plasmids in Lactococcus. lactis subsp. lactis. Appl. Environ. Microbiol. 57:539–48 Froseth BR, Harlander SK, McKay LL. 1988a. Plasmid-mediated reduced phage sensitivity in Streptococcus lactis KR5. J. Dairy Sci. 71:275–84 10.14

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Froseth BR, Herman RE, McKay LL. 1988b. Cloning of nisin resistance and replication origin on 7.6-kilobase EcoRI fragment of pNP40 from Streptococcus lactis subsp. diacetylactis DRC3. Appl. Environ. Microbiol. 54:2136–39 Froseth BR, McKay LL. 1991. Development and application of pFM011 as a possible food-grade cloning vector. J. Dairy Sci. 74:1445–53 Gasson MJ, Davies FL. 1980. High-frequency conjugation associated with Streptococcus lactis donor cell aggregation. J. Bacteriol. 143:1260–64 Guerry P, LeBlanc DJ, Falkow S. 1973. General method for the isolation of plasmid deoxyribonucleic acid. J. Bacteriol. 116:1064–66 Hengstenberg WJ, Egan JB, Morse ML. 1967. Carbohydrate transport in Staphylococcus aureus. V. The accumulation of phosphorylated carbohydrate derivatives and evidence for a new enzyme splitting lactose phosphate. Proc. Nat. Acad. Sci. USA 58:274–79 Herman RE, McKay LL. 1986. Cloning and expression of the β-D-galactosidase gene from Streptococcus thermophilus in Escherichia coli. Appl. Environ. Microbiol. 52:45–50 Hirsch A. 1952. The evolution of the lactic streptococci. J. Dairy Res. 19:290–93 Horng JS, Polzin KM, McKay LL. 1991. Replication and temperature-sensitive maintenance functions of the lactose plasmid pSK11L from Lactococcus lactis subsp. cremoris. J. Bacteriol. 173:7573–81 Kempler GM, McKay LL. 1979a. Characterization of plasmid deoxyribonucleic acid in Streptococcus lactis subsp. diacetylactis: evidence for plasmid linked citrate utilization. Appl. Environ. Microbiol. 37:316–23 Kempler GM, McKay LL. 1979b. Genetic evidence for plasmid-linked lactose metabolism in Streptococcus lactis subsp. diacetylactis. Appl. Environ. Microbiol. 37:1041–43 Kempler GM, McKay LL. 1980. An improved medium for the detection of citrate fermenting Streptococcus lactis subsp. diacetylactis. Appl. Environ. Microbiol. 39:926–27 Klaenhammer TR, McKay LL, Baldwin KA. 1978. Improved lysis of group N streptococci for isolation and rapid characterization of plasmid deoxyribonucleic acid. Appl. Environ. Microbiol. 35:592–600 Kondo JK, McKay LL. 1982. Transformation of Streptococcus lactis protoplasts by plasmid deoxyribonucleic acid. Appl. Environ. Microbiol. 43:1213–15 Kondo JK, McKay LL. 1984. Plasmid transformation of Streptococcus lactis protoplasts: optimization and use in molecular cloning. Appl. Environ. Microbiol. 48:252–59 Krishnaswamy MA, Babel FJ. 1951. Biacetyl production by cultures of lactic acid-producing streptococci. J. Dairy Sci. 34:374–78 Kuhl SA, Larsen LD, McKay LL. 1979. Plasmid profiles of lactose-negative and proteinase-deficient mutants of Streptococcus lactis C10, ML3 and M18. Appl. Environ. Microbiol. 37:1193–95 Leenhouts K, Kok J, Venema G. 1990. Stability of integrated plasmids in the chromosome of Lactococcus lactis. Appl. Environ. Microbiol. 56:2726–35 Laible NJ, Rule PL, Harlander SK, McKay LL. 1987. Identification and cloning of plasmid DNA coding for abortive phage infection from Streptococcus lactis subsp. diacetylactis. J. Dairy Sci. 70:2211–19 Lambowitz AM, Zimmerly S. 2004. Mobile group II introns. Ann. Rev. Genet. 38:1–35 Larsen LD, McKay LL. 1978. Isolation and characterization of plasmid deoxyribonucleic acid in Streptococcus cremoris. Appl. Environ. Microbiol. 36:944–52 McIntyre DA, Harlander SK. 1989. Improved electroporation efficiency of intact Lactococcus lactis subsp. lactis cells grown in defined media. Appl. Environ. Microbiol. 55:2621–26 McKay LL, Baldwin KA. 1973. Induction of prophage in Streptococcus lactis C2 by ultraviolet irradiation. Appl. Microbiol. 25:682–84 McKay LL, Baldwin KA. 1974a. Altered metabolism in a Streptococcus lactis C2 mutant deficient in lactic dehydrogenase. J. Dairy Sci. 57:181–86 McKay LL, Baldwin KA. 1974b. Simultaneous loss of proteinase and lactose utilizing enzyme activities in Streptococcus lactis and reversal of loss by transduction. Appl. Microbiol. 28:342–46 McKay LL, Baldwin KA. 1975. Plasmid distribution and evidence for a proteinase plasmid in Streptococcus lactis. Appl. Microbiol. 29:546–48 McKay LL, Baldwin KA. 1978. Stabilization of lactose metabolism in Streptococcus lactis C2. Appl. Environ. Microbiol. 36:360–67 www.annualreviews.org • An Amazing Journey


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McKay LL, Baldwin KA. 1984. A conjugative 40 megadalton plasmid in Streptococcus lactis subsp. diacetylactis DRC3 is associated with resistance to nisin and bacteriophage. Appl. Environ. Microbiol. 47:68–74 McKay LL, Baldwin KA, Efstathiou JD. 1976. Transductional evidence for plasmid linkage of lactose metabolism in Streptococcus lactis C2. Appl. Environ. Microbiol. 32:45–52 McKay LL, Baldwin KA, Walsh PM. 1980. Conjugal transfer of genetic information in group N streptococci. Appl. Environ. Microbiol. 40:84–91 McKay LL, Baldwin KA, Zottola EA. 1972. Loss of lactose metabolism in lactic streptococci. Appl. Microbiol. 23:1090–96 McKay LL, Bohanon MJ, Polzin KM, Rule PL, Baldwin KA. 1989. Localization of separate genetic loci for reduced sensitivity towards small isometric-headed bacteriophage skl and prolate-headed bacteriophage c2 on pGBK17 from Lactococcus lactis subsp. lactis KR2. Appl. Environ. Microbiol. 55:2702–9 McKay LL, Cords BR, Baldwin KA. 1973. Transduction of lactose metabolism in Streptococcus lactis C2. J. Bacteriol. 115:810–15 McKay LL, Miller A, Sandine WE, Elliker PR. 1970. Mechanisms of lactose utilization by lactic acid streptococci: enzymatic and genetic analyses. J. Bacteriol. 102:804–9 McKay LL, Walter LA, Sandine WE, Elliker PR. 1969. Involvement of phosphoenolpyruvate in lactose utilization by group N streptococci. J. Bacteriol. 99:603–10 McLandsborough LA, Kolaetis KM, Requena T, McKay LL. 1995. Cloning and characterization of the abortive infection genetic determinant abiD isolated from pBF61 of Lactococcus lactis subsp. lactis KR5. Appl. Environ. Microbiol. 61:2023–26 McLandsborough LA, Sechaud L, McKay LL. 1998. Synergistic effects of abiE or abiF from pNP40 when cloned in combination with abiD from pBF61. J. Dairy Sci. 81:362–68 Mills DA, Manias DA, McKay LL, Dunny GM. 1997. Homing of a group II intron from Lactococcus lactis subsp. lactis ML3. J. Bacteriol. 179:6107–11 Mills DA, McKay LL, Dunny GM. 1996. Splicing of a Group II intron involved in the conjugative transfer of pRS01 in lactococci. J. Bacteriol. 178:3531–38 Mills DA, Phister TG, Dunny GM, McKay LL. 1998. An origin of transfer (oriT) on the conjugative element pRS01 from Lactococcus lactis subsp. lactis ML3. Appl. Environ. Microbiol. 64:1541–42 Murphy MC, Steele JL, Daly C, McKay LL. 1988. Concomitant conjugal transfer of reduced bacteriophage sensitivity mechanisms with lactose and sucrose fermenting ability in lactic streptococci. Appl. Environ. Microbiol. 54:1951–56 Park C, McKay LL. 1975. Induction of prophage in lactic streptococci isolated from commercial dairy starter cultures. J. Milk Food Technol. 38:594–97 Polzin KM, McKay LL. 1991. Identification, DNA sequence, and distribution of IS981, a new, high copy number insertion sequence in lactococci. Appl. Environ. Microbiol. 57:734–43 Polzin KM, McKay LL. 1992. Development of a lactococcal integration vector by using IS981 and a temperature-sensitive lactococcal replication region. Appl. Environ. Microbiol. 58:476–84 Polzin KM, Shimizu-Kadota. 1987. Identification of a new insertion element similar to gram-negative IS26, on the lactose plasmid of Streptococcus lactis ML3. J. Bacteriol. 169:5481–84 Riepe HR, McKay LL. 1994. Oversecretion of the neutral protease from Bacillus subtilis in Lactococcus lactis spp. lactis JF254. J. Dairy Sci. 77:2150–59 Riepe HR, Pillidge CJ, Gopal PK, McKay LL. 1997. Characterization of the highly autolytic Lactococcus lactis subsp. cremoris strains CO and 2250. Appl. Environ. Microbiol. 63:3757–63 Sandine WE, Elliker PR, Anderson AW. 1957. A simple apparatus for measurement of gas production and activity of lactic starter cultures. Milk Prod. J. 48:12–15 Scherwitz KM, Baldwin KA, McKay LL. 1983. Plasmid linkage of a bacteriocin-like substance in Streptococcus lactis subsp. diacetylactis strain WM4: transferability to Streptococcus lactis. Appl. Environ. Microbiol. 45:1506– 8 Scherwitz-Harmon KM, McKay LL. 1987. Restriction enzyme analysis of lactose and bacteriocin plasmids from Streptococcus lactis subsp. diacetylactis WM4 and cloning of Bc1I fragments coding for bacteriocin production. Appl. Environ. Microbiol. 53:1171–74 Snook RJ, McKay LL. 1981. Conjugal transfer of lactose-fermenting ability among Streptococcus cremoris and Streptococcus lactis. Appl. Environ. Microbiol. 43:904–11


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Snook RJ, McKay LL, Ahlstrand GG. 1981. Transduction of lactose metabolism by Streptococcus cremoris C3 temperate phage. Appl. Environ. Microbiol. 43:897–903 Steele JL, McKay LL. 1989. Conjugal transfer of genetic material in lactococci: a review. J. Dairy Sci. 72:3388– 97 Steele JL, Murphy MC, Daly C, McKay LL. 1989. DNA-DNA homology among lactose- and sucrosefermenting transconjugants from Lactococcus lactis strains exhibiting reduced bacteriophage sensitivity. Appl. Environ. Microbiol. 55:2410–13 Stoddard GW, Richardson GH. 1986. Effect of proteolytic activity of Streptococcus cremoris on cottage cheese yields. J. Dairy Sci. 69:9–14 Stoddard GW, Petzel JP, Van Belkum MN, Kok J, McKay LL. 1992. Molecular analyses of the Lactococcin A gene cluster from Lactococcus lactis subsp. lactis biovar diacetylactis WM4. Appl. Environ. Microbiol. 58:1952– 61 Tynkkynen S, Buist G, Kunji E, Kok J, Poolman B, et al. 1993. Genetic and biochemical characterization of the oligopeptide transport system of Lactococcus lactis. J. Bacteriol. 175:7523–32 van de Guchte MJ, Kodde J, van der Vossen JMBM, Kok J, Venema G. 1990. Heterologous gene expression in Lactococcus lactis subsp. lactis: synthesis, secretion, and processing of the Bacillus subtilis neutral protease. Appl. Environ. Microbiol. 56:2606–11 Walsh PM, McKay LL. 1981. Recombinant plasmid associated with cell aggregation and high-frequency conjugation of Streptococcus lactis ML3. J. Bacteriol. 146:937–44 Wang H, Yu W, Coolbear T, O’Sullivan D, McKay LL. 1998. A deficiency in aspartate biosynthesis in Lactococcus lactis subsp. lactis C2 causes slow milk coagulation. Appl. Environ. Microbiol. 64:1673–79 Yu W, Gillies K, Kondo JK, Broadbent JR, McKay LL. 1996. Loss of plasmid-mediated oligopeptide transport system in lactococci: another reason for slow milk coagulation. Plasmid 35:145–55



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