@article{callanan_russell_klaenhammer_2007, title={Modification of Lactobacillus beta-glucuronidase activity by random mutagenesis}, volume={389}, ISSN={["0378-1119"]}, DOI={10.1016/j.gene.2006.10.022}, abstractNote={The Lactobacillus gasseri ADH beta-glucuronidase gene, gusA, was cloned previously and found to exhibit excellent activity in acidic pH ranges, with maximal activity at pH 5.0. In contrast, activity was limited in neutral pH ranges of 6-7. In an effort to improve the activity of the reporter enzyme in neutral pH ranges, the gusA gene was cloned into the broad host range vector, pGK12, and subjected to random mutagenesis by passage through Epicurian coli mutator strain XL1-Red. Two mutant alleles, gusA2 and gusA3, were recovered that produced beta-glucuronidase with increased activity in neutral pH ranges. One of these, gusA3, was significantly more active in the pH range of 4-8 in both Escherichia coli and L. gasseri. Sequence analysis of gusA2 and gusA3 revealed single base pair changes that resulted in D524G and D573A substitutions, respectively. The modified GusA3 enzyme has expanded potential for use as a reporter enzyme in expression hosts that are not acidophilic, as well as lactic acid bacteria and other microorganisms that grow in acidifying environments.}, number={2}, journal={GENE}, author={Callanan, Michael J. and Russell, William M. and Klaenhammer, Todd R.}, year={2007}, month={Mar}, pages={122–127} }
 @article{altermann_russell_azcarate-peril_barrangou_buck_mcauliffe_souther_dobson_duong_callanan_et al._2005, title={Complete genome sequence of the probiotic lactic acid bacterium Lactobacillus acidophilus NCFM}, volume={102}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.0409188102}, abstractNote={Lactobacillus acidophilusNCFM is a probiotic bacterium that has been produced commercially since 1972. The complete genome is 1,993,564 nt and devoid of plasmids. The average GC content is 34.71% with 1,864 predicted ORFs, of which 72.5% were functionally classified. Nine phage-related integrases were predicted, but no complete prophages were found. However, three unique regions designated as potential autonomous units (PAUs) were identified. These units resemble a unique structure and bear characteristics of both plasmids and phages. Analysis of the three PAUs revealed the presence of two R/M systems and a prophage maintenance system killer protein. A spacers interspersed direct repeat locus containing 32 nearly perfect 29-bp repeats was discovered and may provide a unique molecular signature for this organism.In silicoanalyses predicted 17 transposase genes and a chromosomal locus for lactacin B, a class II bacteriocin. Several mucus- and fibronectin-binding proteins, implicated in adhesion to human intestinal cells, were also identified. Gene clusters for transport of a diverse group of carbohydrates, including fructooligosaccharides and raffinose, were present and often accompanied by transcriptional regulators of the lacI family. For protein degradation and peptide utilization, the organism encoded 20 putative peptidases, homologs for PrtP and PrtM, and two complete oligopeptide transport systems. Nine two-component regulatory systems were predicted, some associated with determinants implicated in bacteriocin production and acid tolerance. Collectively, these features within the genome sequence ofL. acidophilusare likely to contribute to the organisms' gastric survival and promote interactions with the intestinal mucosa and microbiota.}, number={11}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, publisher={Proceedings of the National Academy of Sciences}, author={Altermann, E and Russell, WM and Azcarate-Peril, MA and Barrangou, R and Buck, BL and McAuliffe, O and Souther, N and Dobson, A and Duong, T and Callanan, M and et al.}, year={2005}, month={Mar}, pages={3906–3912} }
 @article{tuler_callanan_klaenhammer_2002, title={Overexpression of peptidases in Lactococcus and evaluation of their release from leaky cells}, volume={85}, ISSN={["1525-3198"]}, DOI={10.3168/jds.S0022-0302(02)74326-9}, abstractNote={Walker and Klaenhammer (2001) developed a novel expression system in Lactococcus lactis that facilitated the release of beta-galactosidase (117 kDa monomer) without the need for secretion or export signals. The system is based on the controlled expression of integrated prophage holin and lysin cassettes via a lactococcal bacteriophage phi31 transcriptional activator (Tac31A) that resides on a high-copy plasmid. Approximately 85% of beta-galactosidase activity was detected in the supernatant of leaky lactococci without evidence of hindered growth, cell lysis, or membrane damage. The objective of this study was to determine if intracellular peptidases were externalized from leaky lactococci. Five L. lactis peptidases (PepA, PepC, PepN, PepO and PepXP) and two Lactobacillus helveticus peptidases (PepN and PepO) were cloned and overexpressed on two high-copy vectors. The lactococcal peptidases were also cloned into the high-copy vector that contained the Tac31A transcriptional activator to determine if they were externalized from the leaky prophage-containing L. lactis subsp. lactis strain NCK203. Two of the lactococcal peptidases (PepA and PepO) required an additional strong promoter (Lactobacillus paracasei P144) and optimized assay conditions to detect enzyme activity. Results showed different levels of enzymatic overexpression associated with the cellular fraction (2 to 250-fold increases in activity) and negligible amounts of activity present within the supernatant fraction (0 to 6% of total peptidase activity). The lactococcal phage-based protein release mechanism did not facilitate the externalization of the lactococcal peptidases investigated in this study.}, number={10}, journal={JOURNAL OF DAIRY SCIENCE}, author={Tuler, TR and Callanan, MJ and Klaenhammer, TR}, year={2002}, month={Oct}, pages={2438–2450} }
 @article{callanan_pw o'toole_lubbers_polzin_2001, title={Examination of lactococcal bacteriophage c2 DNA replication using two-dimensional agarose gel electrophoresis}, volume={278}, ISSN={["0378-1119"]}, DOI={10.1016/S0378-1119(01)00702-8}, abstractNote={The ori locus of the prolate-headed lactococcal bacteriophage c2 supports plasmid replication in Lactococcus lactis in the absence of phage infection. To determine whether phage c2 DNA replication is initiated at the ori locus in vivo and to investigate the mechanism of phage DNA replication, replicating intermediates of phage c2 were analyzed using neutral/neutral two-dimensional agarose gel electrophoresis (2D). The 2D data revealed that c2 replicates via a theta mechanism and localized the initiation of theta replication to the ori region of the c2 genome.}, number={1-2}, journal={GENE}, author={Callanan, MJ and PW O'Toole and Lubbers, MW and Polzin, KM}, year={2001}, month={Oct}, pages={101–106} }