Progress 10/01/00 to 09/30/06

Outputs
The lactic acid bacteria (LAB) are functionally related group of organisms known primarily for their bioprocessing roles in food and beverages. More recently, select members of the LAB have been implicated in a number of probiotic roles that impact general health and well being. Genomic analyses of multiple members of the LAB, at the genus, species, and strain level, have now elucidated many genetic features that direct their fermentative and probiotic roles. This information is providing an important platform for understanding core mechanisms that control and regulate bacterial growth, survival, signaling, and fermentative processes and, in some cases, potentially underlying probiotic activities within complex microbial and host ecosystems. We have successfully identified genes that correlate with the following important phenotypes: acid tolerance, oxalate degradation, bile salt hydrolase activity, proteolytic ability, utilization of fructo-oligosaccharides, and adhesion to intestinal epithelial cells.

Impacts
The availability of genome information and functional genomic analyses has begun to reveal the mechanisms through which probiotic bacteria function. Features identified include those which promote survival through the stomach, passage through the GI track, attachment to epithelial cells, tolerance to bile and acid, and utilization of carbohydrates that promote in-vivo competitiveness. With this accumulating knowledge, it now becomes possible identify the most effective and functional probiotic cultures for use in food and dietary adjuncts and promote the expression of desirable gene sets for important probiotic features.

Publications

• Azcarate-Peril, M.A., E. Altermann, R.L. Hoover-Fitzula, R.J. Cano, and T.R. Klaenhammer. 2004. Identification and inactivation of genetic loci involved with Lactobacillus acidophilus acid tolerance. Appl. Environ. Microbiol. 70:5315-5322.
• Altermann, E., B.L. Buck, R. Cano, and T.R. Klaenhammer. 2004 Identification and phenotypic characterization of the cell-division protein CdpA. Gene 342:189-197.
• Duong, T., R. Barrangou, W.M. Russell, and T.R. Klaenhammer. 2005. Characterization of the tre Locus and Analysis of Trehalose Cryoprotection in Lactobacillus acidophilus NCFM. Appl. Environ. Microbiol. 72: 1218-1225.
• Altermann, E., and T.R. Klaenhammer. 2005. PathwayVoyager: pathway mapping using the Kyoto Encyclopedia of genes and genomes (KEGG) database. BMC Genomics 6:60.
• Klaenhammer, T.R., deVos, W.M., and Mercenier, A. 2005. Genomics of probiotic lactic acid bacteria: impacts on functional foods. In Bioprocesses and biotechnology for functional foods and utraceuticals. Marcel Dekker, NY, pp 63-78.
• Russell, W.M., and T.R. Klaenhammer. 2001. An efficient system for directed integration into the Lactobacillus acidophilus and Lactobacillus gasseri chromosome via homologous recombination. Appl. Environ. Microbiol. 67:4361-4364.
• Barrangou, R., E. Altermann, R. Hutkins, R. Cano, and T. Klaenhammer. 2003. Functional and comparative genomic analyses of an operon involved in fructooligosaccharide utilization by Lactobacillus acidophilus. Proc. Nat. Acad. Sci. USA. 100: 8957-8962.
• Reid, G., Sanders, M.E., Gaskins, H.R., Gibson, G.R., Mercenier, A., Rastall, R., Roberfroid, M., Rowland, I., Cherbut, C., and Klaenhammer, T.R. 2003. New scientific paradigms for probiotics and prebiotics. J. Clin. Gastroenterology 37:105-118.
• Altermann, E., and T.R. Klaenhammer. 2003. GAMOLA: a new local solution for sequence annotation and analyzing draft and finished prokayotic genomes. OMICS 7:161-169.
• Pridmore, R.D., B. Berger, F. Desiere D. Vilanova, C. Barretto, A.-C.Pittet, M.-C. Zwahlen, M. Rouvet, E. Altermann, R. Barrangou, B. Mollet, A. Mercenier, T.R. Klaenhammer, F. Arigoni and M.A. Schell. 2004. The genome Sequence of the Probiotic Intestinal Bacterium Lactobacillus johnsonii NCC 533. Proc. Nat. Acad. Sci U.S.A. 101: 2512 2517
• Azcarate-Peril, M.A., J.M. Bruno-Barcena, H.M. Hassan and T.R. Klaenhammer. 2006. Transcriptional and Functional Analysis of Oxalyl-Coenzyme A (CoA) Decarboxylase and Formyl-CoA Transferase Genes from Lactobacillus acidophilus. Appl. Environ. Microbiol. 72:1891-1899.
• Ventura M, Canchaya C, Bernini V, Altermann E, Barrangou R, McGrath S, Claesson MJ, Li Y, Leahy S, Walker CD, Zink R, Neviani E, Steele J, Broadbent J, Klaenhammer TR, Fitzgerald GF, O'toole PW, van Sinderen D. 2006. Comparative genomics and transcriptional analysis of prophages identified in the genomes of Lactobacillus gasseri, Lactobacillus salivarius, and Lactobacillus casei. Appl Environ Microbiol. 72:3130 3146.
• Makarova K, Slesarev A, Wolf Y, Sorokin A, Mirkin B, Koonin E, Pavlov A, Pavlova N, Karamychev V, Polouchine N, Shakhova V, Grigoriev I, Lou Y, Rohksar D, Lucas S, Huang K, Goodstein DM, Hawkins T, Plengvidhya V, Welker D, Hughes J, Goh Y, Benson A, Baldwin K, Lee JH, Diaz-Muniz I, Dosti B, Smeianov V, Wechter W, Barabote R, Lorca G, Altermann E, Barrangou R, Ganesan B, Xie Y, Rawsthorne H, Tamir D, Parker C, Breidt F, Broadbent J, Hutkins R, O'Sullivan D, Steele J, Unlu G, Saier M, Klaenhammer T, Richardson P, Kozyavkin S, Weimer B, Mills D. 2006. Comparative genomics of the lactic acid bacteria. Proc Natl Acad Sci U S A. 103:15611-15616.

Progress 10/01/04 to 09/30/05

Outputs
Among many important features of probiotic cultures are those abilities that promote survival and passage of the organism through the stomach and into the gastrointestinal tract. Two-component regulatory systems are one primary mechanism for environmental sensing and signal transduction. Annotation of the complete genome sequence of the probiotic bacterium Lactobacillus acidophilus NCFM revealed nine two-component regulatory systems. In this study, the histidine protein kinase of a two-component regulatory system (LBA1524HPK-LBA1525RR), similar to the acid-related system lisRK from Listeria monocytogenes, was insertionally inactivated. A whole-genome microarray containing 97.4% of the annotated genes of L. acidophilus was used to compare genome-wide patterns of transcription at various pHs between the parent and the histidine protein kinase mutant. The expression pattern of approximately 80 genes was affected by the LBA1524HPK mutation. Putative LBA1525RR target loci included two oligopeptide-transport systems present in the L. acidophilus genome. The mutant exhibited lower tolerance to acid and ethanol in logarithmic-phase cells and poor acidification rates in milk. Supplementation of milk with casamino acids essentially restored the acid-producing ability of the mutant, providing additional evidence for a role of this two component system in regulating proteolytic activity in L. acidophilus. Tolerance to the presence of bile is another attribute that allows L. acidophilus to survive within this environment. Early log phase cells (OD600 = 0.3) of L. acidophilus were exposed to 5% Oxgall (pH<5.5) in MRS for 30 minutes, and the resulting transcriptional responses were analyzed using the whole genome microarray. Twenty-seven genes were shown to be induced specifically by bile. Among them, the genes in a putative operon (LBA1427 to LBA1432), composed of a two-component regulatory system (2CRS) and four poorly characterized genes, were significantly (P<0.01) induced by bile. Inactivation of the LBA1430 (a predicted histidine kinase, HPK) was accomplished by targeted plasmid insertion via homologous recombination. The growth rates of the HPK mutant and a control strain were compared in MRS supplemented with 0%, 0.3%, 0.5% and 1.0% Oxgall. The growth rate for the HPK mutant was reduced significantly in the presence of 0.3% and 0.5% Oxgall. The results indicate that L. acidophilus relies on two-component regulatory systems to regulate the expression of genes that contribute significantly to acid tolerance, proteolytic activity and bile tolerance.

Impacts
The availability of genome information and confirmation of microbial functions by functional genomic analyses has begun to reveal the mechanisms through which probiotic bacteria function. Features identified include those which promote survival through the stomach, passage through the GI track, attachment to epithelial cells, tolerance to bile and acid, and utilization of carbohydrates that promote in-vivo competitiveness. With this accumulating knowledge, it now becomes identify the most effective and functional probiotic cultures for use in food and dietary adjuncts and promote expression of desirable gene sets.

Publications

• Altermann, E. , W. M. Russell, M.A.Azcarate-Peril, R. Barrangou, B.L.Buck, O. McAuliffe, N. Souther, A. Dobsen, T. Doung, M. Callanan, S. Lick, A. Hamrick, R. Cano, and T.R. Klaenhammer. 2005. Complete genome sequence of the probiotic lactic acid bacterium Lactobacillus acidophilus NCFM. Proc. Nat. Acad. Sci. USA 102: 3906-3912.
• Mohamadzadeh, M., S. Olson, W.V. Kalina, G. Ruthel, G.L. Demmin, K.L. Warfield, S.Bavari, and T. R. Klaenhammer. 2005. Lactobacilli activate human dendritic cells that skew T cells toward T helper 1 polarization. Proc. Nat. Acad. Sci. USA. 102:2280-2285.
• Rastall, R.A., G.R. Gibson, H.S. Gill, F. Guarner, T. R. Klaenhammer, B. Pot, G. Reid, I.R. Rowland, M.E. Sanders. (2005). Modulation of the microbial ecology of the human colon by probiotics, prebiotics and synbiotics to enhance human health: An overview of enabling science and potential applications. FEMS Microbiology Ecology 52: 145-152.
• Klaenhammer, T.R., A. Azcarate Peril, R. Barrangou, T. Doung and E. Altermann. 2005. Genomic perspectives on probiotic lactic acid bacteria. Bioscience Microflora 24:31-33.
• Klaenhammer, T.R., R. Barrangou, B.L. Buck, M.A. Azcarate-Peril, E.Altermann. 2005. Genomic features of lactic acid bacteria effecting bioprocessing and health. FEMS Microbiology Reviews. 29:393-409.
• McAuliffe, O., R. J. Cano, and T.R. Klaenhammer. 2005. Genetic analysis of two bile salt hydrolase activities in Lactobacillus acidophilus NCFM. Appl. Environ. Microbiol. 71:4925-4929.
• Azcarate-Peril, M.A., O. McAuliffe, E. Altermann, S.Lick, W. M. Russell, and T.R. Klaenhammer. 2005. Microarray analysis of a two-component regulatory system involved in acid resistance and proteolytic activity in Lactobacillus acidophilus. Appl. Envir. Microbiol. 71: 5794-5804.
• Logan Buck, Eric Altermann, Tina Svingerud, and Todd R. Klaenhammer. 2005. Functional analysis of putative adhesion factors in Lactobacillus acidophilus NCFM. Appl. Envir. Microbiol. 2005 71: 8344-8351.

Progress 10/01/03 to 09/30/04

Outputs
Lactic acid bacteria are widely used in commercial fermentation processes and as probiotics. Lactobacillus acidophilus is an important member of the human and infant gastrointestinal tract and has been widely used as a probiotic culture delivered in dairy foods and dietary supplements. The genome of L. acidophilus NCFM has been completely sequenced and automatically annotated. A whole-genome microarray was constructed by printing cDNA PCR amplicons of 1,897 genomic ORFs. RNA samples were isolated from mid-log cells grown in broth containing different carbohydrates, namely glucose, fructose, galactose, lactose, sucrose, raffinose and fructo-oligosaccharides. Microarray data was analyzed using mixed models of analysis of variance. Specifically-induced genes and operons included PTS and ABC transporters, as well as carbohydrate hydrolases likely involved in transfer and catabolism of carbohydrates. Genes encoding enzymes involved in central energy metabolism, specifically glycolysis, were consistently highly expressed, regardless of the carbohydrate source. Additionally, analysis of the promoter-operator region of genes specifically induced revealed the common presence of catabolite response elements, suggesting regulation of gene expression via carbon catabolite repression. A combined approach of in silico and gene expression analysis was also used to identify potentially highly expressed genes under varying stress conditions (acid, bile, ethanol, and ammonium oxalate). Microarray data were used to analyze changes in codon usage and codon adaptation indices based on highly expressed and induced gene sets. Indices calculated on highly expressed genes showed a significantly lower overall adaptation index (0.3) relative to indices based on induced genes (0.44). The results indicated that codon adaptation in L. acidophilus has evolved to ensure a more efficient translation under stress conditions.

Impacts
The availability of genome information and confirmation of microbial functions by functional genomic analyses has begun to reveal the mechanisms through which probiotic bacteria function. Features identified include those which promote survival through the stomach, passage through the GI track, attachment to mucin-secreting epithelial cells, and utilization of carbohydrates that promote in-vivo competitiveness. With this accumulating knowledge, it now becomes feasible select and identify the most effective and functional probiotic cultures for use in food and dietary adjuncts.

Publications

• Ventura, M., C. Canchaya, V. Meylan, T.R. Klaenhammer, and R. Zink. 2003. Analysis, characterization, and loci of the tuf genes in Lactobacillus and Bifidobacterium species and their direct application for species evaluation. Appl. Environ. Microbiol. 69:6908-6922.
• Azcarate-Peril, M.A., E. Altermann, R.L. Hoover-Fitzula, R.J. Cano, and T.R. Klaenhammer. 2004. Identification and inactivation of genetic loci involved with Lactobacillus acidophilus acid tolerance. Appl. Environ.Microbiol. 70:5315-5322..
• Altermann, E., B.L. Buck, R. Cano, and T.R. Klaenhammer. 2004. Identification and phenotypic characterization of the cell-division protein CdpA. Gene 342:189-197.
• Pridmore, R.D., B. Berger, F. Desiere D. Vilanova, C. Barretto, A.-C.Pittet, M.-C. Zwahlen, M. Rouvet, E. Altermann, R. Barrangou, B. Mollet, A. Mercenier, T.R. Klaenhammer, F. Arigoni and M.A. Schell. 2004. The genome Sequence of the Probiotic Intestinal Bacterium Lactobacillus johnsonii NCC 533. Proc. Nat. Acad. Sci U.S.A. 101: 2512-2517
• Majhenic, A.C., K. Venema, G.E. Allison, B.B. Matijasic, I. Rogelj, and T.R. Klaenhammer. 2004. DNA analysis of the genes encoding acidocin LF221 A and acidocin LF221 B, two bacteriocins producted by Lactobacillus gasseri LF221. Appl. Microbiol. Biotechnol. 63: 705-714.

Progress 10/01/02 to 09/30/03

Outputs
Lactic acid bacteria are widely used in commercial fermentation processes and as probiotics. Lactobacillus acidophilus is an important member of the human and infant gastrointestinal tract and has been widely used as probiotic culture delivered in dairy foods and dietary supplements. The genome of L. acidophilus NCFM was completely sequenced and automatically annotated. Manual annotation and genome polishing were carried out and revealed the presence of 1965 open reading frames (ORFs) resulting in a coding percentage of 89 %. The average %guanine + cytosine content was 34.71. The deduced proteins were classified according to the Pfam or COG roles. Searching the complete genome revealed regions that were targeted for detailed functional analysis. Notable areas included putative adherence proteins and regions encoding carbohydrate transport and catabolism. Insertional inactivation of selected ORFs in the genome have confirmed roles for mucin-binding proteins, S-layer proteins, and fibronectin binding proteins in the attachment of the bacterium to Caco-2 epithelial cells, in vitro. Uptake of fructo-oligosaccharides(FOS), a prebiotic, was confirmed to occur via a novel operon encoding an ABC transporter and a fructosidase that hydrolyzes oligofructose moieties intracellularly. Whole genome microarrays were also carried out on log phase cells grown on FOS. The seven genes of the FOS operon were highly induced, as were genes encoding enzymes involved in glycolysis and central energy metabolism. The promoter-operator region of the FOS operon revealed the presence of a catabolite response element, indicating regulation of gene expression via carbon catabolite repression. Catabolite repression was functionally confirmed by glucose repression, and FOS induction of the operon.

Impacts
The availability of genome information and confirmation of microbial functions by functional genomic analyses has begun to reveal the mechanisms through which probiotic bacteria function. Features identified include those which promote survival through the stomach, passage through the GI track, attachment to mucin-secreting epithelial cells, and utilization of carbohydrates that promote in-vivo competitiveness. With this accumulating knowledge, it now becomes feasible select and identify the most effective and functional probiotic cultures for use in food and dietary adjuncts.

Publications

• Barrangou, R., Altermann, E., Hutkins, R., Cano, R., and Klaenhammer, T.R. 2003. Functional and comparative genomic analyses of an operon involved in fructooligosaccharide utilization by Lactobacillus acidophilus. Proc. Nat. Acad. Sci. USA. 100: 8957-8962.
• Reid, G., Sanders, M.E., Gaskins, H.R., Gibson, G.R., Mercenier, A., Rastall, R., Roberfroid, M., Rowland, I., Cherbut, C., and Klaenhammer, T.R. 2003. New scientific paradigms for probiotics and prebiotics. J. Clin. Gastroenterology 37:105-118.
• Altermann, E., and Klaenhammer, T.R. 2003. GAMOLA: a new local solution for sequence annotation and analyzing draft and finished prokayotic genomes. OMICS 7:161-169.

Progress 10/01/01 to 09/30/02

Outputs
The complete genome of Lactobacillus acidophilus NCFM and eight major contigs representing the Lactobacillus gasseri genome have been sequenced and annotated. A Global Annotation of Multiplexed On-site Blasted DNA-sequences (GAMOLA) software solution was developed and used to identify gene targets for detailed bioinformatic and functional analysis. The NCFM genome is 1,993,598 bp, encoding 1905 genes with an GC-content is 34.7 %. Only 26 % of the predicted ORFs remained without an initial functional assignment. A whole genome DNA microarray has been developed for the L. acidophilus genome using PCR products amplified from 96% of the predicted ORFs. Gene knockouts have been successfully completed for the following: bile salt hydrolases (bshA, bshB), S-layer protein (slpA), beta-fructosidase, and surface proteins suspected in adherence mechanisms. These mutants are currently being evaluated for their phenotypic characteristics. Comparative and functional genomic analyses are ongoing.

Impacts
Demonstration of critical genetic features that are responsible for the growth, survival and beneficial activites of probiotic cultures in foods and the human gastrointestinal tract.

Publications

• Klaenhammer, T., E. Altermann, F. Arigoni, A. Bolotin, F. Breidt, J. Broadbent, R. Cano, S. Chaillou, J. Deutscher, M. Gasson, M. van de Guchte, J. Guzzo, A. Hartke, T. Hawkins, P. Hols, R. Hutkins, M. Kleerebezem, J. Kok, O. Kuipers, M. Lubbers, E. Maguin, L. McKay, D. Mills, A. Nauta, R. Overbeek, H. Pel, D. Pridmore, M. Saier, D. van Sinderen, A. Sorokin, J. Steele, D. O?Sullivan, W. de Vos, B. Weimer, M. Zagorec, and R. Siezen. 2002. Discovering lactic acid bacteria by genomics. Antonie van Leeuwenhoek 82: 59-71.
• Yother, J. P. Trieu-Cuot, T.R. Klaenhammer, and W. M. de Vos. 2002. Genetics of Streptococci, Lactococci, and Enterococci: Review of the Sixth International Conference J. Bacteriol. 2002 184: 6085-6092.
• McAuliffe, O., and T.R. Klaenhammer. 2002. Genomic perspectives on probiotics and the gastrointesinal microflora. pp. 263-310. In Probiotics and Prebiotics: Where are we going? G. Tannock (ed.) Caister Academic Press, Norfolk, England.

Progress 10/01/00 to 09/30/01

Outputs
Whole genome sequencing of Lactobacillus acidophilus NCFM and Lactobacillus gasseri ATCC33323 (has been completed in 2001. Functional analysis of the genomes revealed a number of determinants that are likely to be important for probiotic functionality. Among these were genes encoding two bile salt hydrolases, operons for utilization of fructo-oligosaccharides, and numerous stress responsive operons inducible by temperature, pH, and hydrogen peroxide. Genetic knockout strategies were initiated to investigate the roles and functions of a number of these determinants. An efficient method was developed for generation of site-specific chromosomal integrations in thermophilic Lactobacillus species. The strategy is an adaptation of the lactococcal pORI system and relies on the simultaneous use of two plasmids, one temperature sensitive plasmid providing repA in trans for a second insertion vector encoding the targeted region of homology. The functionality of the integration strategy was demonstated by the insertional inactivation of the Lactobacillus acidophilus NCFM lacL gene encoding b-galactosidase and the Lactobacillus gasseri ADH gusA gene, encoding b-glucuronidase A. Both comparative and functional genomic analyses are ongoing.

Impacts
Demonstration of critical genetic elements that are responsible for the survival and beneficial activities of probiotic cultures in the human gastrointestinal tract.

Publications

• Russell, W.M. and T.R. Klaenhammer. 2001. Identification and cloning of gusA, encoding a new B-glucuronidase from Lactobacillus gasseri ADH. Appl. Environ. Microbiol. 67: 1253-1267.
• Russell, W.M., and T.R. Klaenhammer. 2001. An efficient system for directed integration into the Lactobacillus acidophilus and Lactobacillus gasseri chromosome via homologous recombination. Appl. Environ. Microbiol. 67:4361-4364.
• Sanders, M.E. and T. R. Klaenhammer. 2001. The Scientific Basis of Lactobacillus acidophilus NCFM Functionality as a Probiotic. J. Dairy Sci. 84:319-331.
• Klaenhammer, T. R. 2001. Probiotics and Prebiotics. Chapter 39, pp. 797-811. In Doyle, M., Beauchat, L, and Montville, T (eds.) Food Microbiology: Fundamentals and Frontiers - 2nd Edition. ASM Press. Washington, DC.

Progress 10/01/99 to 09/30/00

Outputs
Lactobacillus acidophilus and related lactobacilli are natural inhabitants of the human gastrointestinal tract and are delivered as health-promoting bacteria in foods and probiotics. Efforts to understand how these bacteria interact with their human host and elucidate the genetic basis of their proposed benefits are hindered by the paucity of critical genetic tools which are routinely available for other organisms. To address this problem, we are developing genetic tools designed specifically for use among these lactobacilli. A modification of a lactococcal integration system for generating site-specific chromosomal integrations has been applied to Lactobacillus. The system utilizes two pWV01-based plasmids with different antibiotic markers. One of these is the integration plasmid pORI28 (kindly provided by J. Kok, University of Groningen), which has been deleted for the gene encoding RepA. The other plasmid, pTRK669, is unstable at thermophilic temperatures (>37C), and is used as a helper plasmid to establish pORI28 by supplying RepA, in trans. Both plasmids can be stably maintained with double antibiotic selection at 37C. Removal of antibiotic selection for pTRK669, in conjunction with an increase in temperature, results in the loss of pTRK669 and selection of cells where pORI28 is integrated into the chromosome. Using this system, the L. acidophilus lacL and L. gasseri gusA genes were successfully disrupted by insertional inactivation using internal regions of homology to direct integration of pORI28. The recovery of integrants in each experiment was 100 percent and integration events were confirmed by PCR, Southern analysis and enzyme assays. This is the first efficient and effective integration strategy developed for thermophilic probiotic lactobacilli such as L. acidophilus and L. gasseri. Because of the broad host range of pWV01 based plasmids, this system is expected to be functional across a broad range of gram-positive bacteria. This system in now being used to direct insertions into genes identified from genome sequencing efforts on L. acidophilus.

Impacts
The food industry in the United States is the country's largest manufacturing industry with revenues exceeding 500 billion dollars per year. One key research priority of this industry is to increase the development of foods that promote health and well being. The functional food category, where probiotics comprise a significant component, is expected to increase this market by over 10 per cent in the next decade. Probiotics are live microorganisms which benefit man or animals by improving their indigenous microflora. Health benefits attributed to probiotics include: maintenance of the normal microflora; pathogen interference, exclusion, and antagonism; immunostimulation; anticarcinogenic and antimutagenic activities; and alleviation of lactose intolerance. However, many of the effects attributed to the ingestion of probiotics remain scientifically unsubstantiated. Today's challenge is to strengthen the science supporting the probiotic concept and establish direct cause and effect mechanisms that are responsible for effects on human health and nutrition. This challenge is being attacked by genomic characterization of probiotic cultures in order to quickly and unequivocally identify key genetic regions that direct intestinal survival, growth, and define the metabolic and physiological processes potentially involved in beneficial roles and activities.

Publications

• Russell, W. M., and T. R. Klaenhammer. 2000. Development of a phospho-alpha-(1,1)-glucosidase, treA, and a novel beta-glucuronidase gene as reporter genes for lactobacilli. Abstracts of the 100th General Meeting of the American Society for Microbiology. Abstract H-106, p372.
• Girgis, H.S., Cano, R.J. and T.R. Klaenhammer. 2000. Tolerance to hydrogen and expression of glutathione reductase in Lactobacillus. IFT Annual Meeting Book of Abstracts. Pp183, Abstract 78D-10.
• Klaenhammer, T.R., and W.M. Russell. 2000. Species of the Lactobacillus acidophilus complex. Encyclopedia of Food Microbiology, Volume 2, pp1151-1157. Robinson, R.K, Batt, C., and Patel, P.D (eds). Academic Press, San Diego
• Kullen, M.J., R.B. Sanozky-Dawes, D.C. Crowell and T.R. Klaenhammer. 2000. Use of DNA sequence of variable regions of the 16SrRNA gene for rapid and accurate identification of bacteria in the Lactobacillus acidophilus complex. J. Appl. Microbiol. 89:511-518.
• Kullen, M.J. and T.R. Klaenhammer.2000. Genetic modification of intestinal lactobacilli and bifidobacteria. Curr. Issues Mol. Biol. 2: 41-50.