Influence of calcium and phosphorus, lactose, and salt-to-moisture ratio on Cheddar cheese quality: changes in residual sugars and water-soluble organic acids during ripening.

Cheddar cheese ripening involves the conversion of lactose to glucose and galactose or galactose-6-phosphate by starter and nonstarter lactic acid bacteria. Under ideal conditions (i.e., where bacteria grow under no stress of pH, water activity, and salt), these sugars are mainly converted to lactic acid. However, during ripening of cheese, survival and growth of bacteria occurs under the stressed condition of low pH, low water activity, and high salt content. This forces bacteria to use alternate biochemical pathways resulting in production of other organic acids. The objective of this study was to determine if the level and type of organic acids produced during ripening was influenced by calcium (Ca) and phosphorus (P), residual lactose, and salt-to-moisture ratio (S/M) of cheese. Eight cheeses with 2 levels of Ca and P (0.67 and 0.47% vs. 0.53 and 0.39%, respectively), lactose at pressing (2.4 vs. 0.78%), and S/M (6.4 vs. 4.8%) were manufactured. The cheeses were analyzed for organic acids (citric, orotic, pyruvic, lactic, formic, uric, acetic, propanoic, and butyric acids) and residual sugars (lactose, galactose) during 48 wk of ripening using an HPLC-based method. Different factors influenced changes in concentration of residual sugars and organic acids during ripening and are discussed in detail. Our results indicated that the largest decrease in lactose and the largest increase in lactic acid occurred between salting and d 1 of ripening. It was interesting to observe that although the lactose content in cheese was influenced by several factors (Ca and P, residual lactose, and S/M), the concentration of lactic acid was influenced only by S/M. More lactic acid was produced in low S/M treatments compared with high S/M treatments. Although surprising for Cheddar cheese, a substantial amount (0.2 to 0.4%) of galactose was observed throughout ripening in all treatments. Minor changes in the levels of citric, uric, butyric, and propanoic acids were observed during early ripening, whereas during later ripening, a substantial increase was observed. A gradual decrease in orotic acid and a gradual increase in pyruvic acid content of the cheeses were observed during 12 mo of ripening. In contrast, acetic acid did not show a particular trend, indicating its role as an intermediate in a biochemical pathway, rather than a final product.

Identification of a gene cluster encoding Krebs cycle oxidative enzymes linked to the pyruvate carboxylase gene in Lactococcus lactis ssp. lactis C2.

We identified a 14-kb pyruvate carboxylase gene-containing fragment from a lactococcal C2-lambda phage genomic library. Downstream of the pyruvate carboxylase gene-containing fragment, a gene cluster coding for open reading frames displaying extensive homology to citrate synthase, aconitase, and a truncated isocitrate dehydrogenase was identified. However, the truncation was shown to have occurred during the cloning by two noncontiguous Sau3AI fragments ligating together. The lactococcal citrate synthase gene consisted of 1323 bp and encoded a 441-amino acid citrate synthase protein. The lactococcal aconitase gene was 2544 bp and encoded an 848-amino acid protein. Corresponding to the complete citrate synthase gene, citrate synthase activity was detected in Lactococcus lactis ssp. lactis C2. Isocitrate dehydrogenase activity was found to be missing in Lactococcus lactis C2, suggesting that the gene may be incomplete or is not expressed, resulting in a requirement for glutamic acid in lactococci.

Classification of a bacterial isolate, from pozol, exhibiting antimicrobial activity against several gram-positive and gram-negative bacteria, yeasts, and molds.

A bacterial isolate, designated CS93, capable of producing a broad-spectrum antimicrobial compound(s) effective against gram-positive and gram-negative bacteria, yeasts, and molds was isolated from pozol, a fermented maize product. This strain was phenotypically similar to another pozol isolate that was previously designated as Agrobacterium azotophilium by other investigators. By using biochemical, phenotypic, and 16S rRNA sequence analysis, both pozol isolates were identified as members of the genus Bacillus, possibly a variant of Bacillus subtilis. While the antimicrobial compound(s) was initially produced only on a solid medium, parameters were identified for production in broth. The compound(s) was heat stable (121 degrees C for 15 min), exhibited activity over a wide pH range (pH 3 to pH 11), and was inactivated by pronase E. The antimicrobial compound(s) was bactericidal and bacteriolytic against Escherichia coli V517, bacteriostatic against Micrococcus luteus, and fungistatic against Saccharomyces cerevisiae. The inhibitory compound(s) could possibly serve as a food biopreservative.

Characterization of AbiR, a novel multicomponent abortive infection mechanism encoded by plasmid pKR223 of Lactococcus lactis subsp. lactis KR2.

The native lactococcal plasmid pKR223 encodes two distinct phage resistance mechanisms, a restriction and modification (R/M) system designated LlaKR2I and an abortive infection mechanism (Abi) which affects prolate-headed-phage proliferation. The nucleotide sequence of a 16,174-bp segment of pKR223 encompassing both the R/M and Abi determinants has been determined, and sequence analysis has validated the novelty of the Abi system, which has now been designated AbiR. Analysis of deletion and insertion clones demonstrated that AbiR was encoded by two genetic loci, separated by the LlaKR2I R/M genes. Mechanistic studies on the AbiR phenotype indicated that it was heat sensitive and that it impeded phage DNA replication. These data indicated that AbiR is a novel multicomponent, heat-sensitive, "early"-functioning Abi system and is the first lactococcal Abi system described which is encoded by two separated genetic loci.

Cloning, sequencing, and expression of the pyruvate carboxylase gene in Lactococcus lactis subsp. lactis C2.

A functional pyc gene was isolated from Lactococcus lactis subsp. lactis C2 and was found to complement a Pyc defect in L. lactis KB4. The deduced lactococcal Pyc protein was highly homologous to Pyc sequences of other bacteria. The pyc gene was also detected in Lactococcus lactis subsp. cremoris and L. lactis subsp. lactis bv. diacetylactis strains.

Group II introns and expression of conjugative transfer functions in lactic acid bacteria.

The homologous lactococcal conjugative elements pRS01 and the sex factor of Lactococcus lactis strain 712 both contain a Group II intron within a gene believed to encode a conjugative relaxase enzyme. This enzyme is responsible for nicking of DNA at the origin of transfer (oriT) sequence of the sex factor DNA to initiate the strand transfer process. Group II introns have been studied in eukaryotes, and several of these elements in yeast mitochondrial genes have received considerable attention. These introns are relatively large in size and generally encode a protein within the intron sequence. In addition to splicing activity. Group II introns are mobile genetic elements. The intron-encoded proteins (IEPs) contain endonuclease and reverse transcriptase domains believed to play an enzymatic role in genetic mobility reactions, while a putative maturase domain is thought to promote splicing by stabilizing the folding of the intron RNA into an active ribozyme structure which carries out the splicing reaction. The lactococcal introns represent the first examples of Group II introns shown to be functional in vivo in prokaryotes. Because of the advantages of a bacterial system for genetic and molecular studies, the Ll.ltrB intron from pRS01 has attracted the attention of several laboratories interested in Group II intron biology. Recently, it has been shown that the system can be adapted to function in Escherichia coli (although at somewhat reduced efficiency). In addition, it has been recently proven that the best studied form of mobility, the homing of the intron into an intronless allele of the cognate exon gene, occurs via an RNA intermediate and does not require DNA homology or generalized host recombination functions. Current efforts are analysis of the role Ll.ltrB splicing in regulating expression of pRS01 conjugation functions. The lactococcal Group II introns represent the first demonstrated genetically mobile prokaryotic retroelements, and they also have considerable potential as genetic engineering tools for Lactic Acid Bacteria (LAB) and other organisms.

Molecular characterization of the Lactococcus lactis LlaKR2I restriction-modification system and effect of an IS982 element positioned between the restriction and modification genes.

The nucleotide sequence of the plasmid-encoded LlaKR2I restriction-modification (R-M) system of Lactococcus lactis subsp. lactis biovar diacetylactis KR2 was determined. This R-M system comprises divergently transcribed endonuclease (llaKR2IR) and methyltransferase (llaKR2IM) genes; located in the intergenic region is a copy of the insertion element IS982, whose putative transposase gene is codirectionally transcribed with llaKR2IM. The deduced sequence of the LlaKR2I endonuclease shared homology with the type II endonuclease Sau3AI and with the MutH mismatch repair protein, both of which recognize and cleave the sequence 5' GATC 3'. In addition, M. LlaKR2I displayed homology with the 5-methylcytosine methyltransferase family of proteins, exhibiting greatest identity with M. Sau3AI. Both of these proteins shared notable homology throughout their putative target recognition domains. Furthermore, subclones of the native parental lactococcal plasmid pKR223, which encode M. LlaKR2I, all remained undigested after treatment with Sau3AI despite the presence of multiple 5' GATC 3' sites. The combination of these data suggested that the specificity of the LlaKR2I R-M system was likely to be 5' GATC 3', with the cytosine residue being modified to 5-methylcytosine. The IS982 element located within the LlaKR2I R-M system contained at its extremities two 16-bp perfect inverted repeats flanked by two 7-bp direct repeats. A perfect extended promoter consensus, which represented the likely original promoter of the llaKR2IR gene, was shown to overlap the direct repeat sequence on the other side of IS982. Specific deletion of IS982 and one of these direct repeats via a PCR strategy indicated that the LlaKR2I R-M determinants do not rely on elements within IS982 for expression and that the efficiency of bacteriophage restriction was not impaired.

A deficiency in aspartate biosynthesis in Lactococcus lactis subsp. lactis C2 causes slow milk coagulation.

A mutant of fast milk-coagulating (Fmc+) Lactococcus lactis subsp. lactis C2, designated L. lactis KB4, was identified. Although possessing the known components essential for utilizing casein as a nitrogen source, which include functional proteinase (PrtP) activity and oligopeptide, di- and tripeptide, and amino acid transport systems, KB4 exhibited a slow milk coagulation (Fmc-) phenotype. When the amino acid requirements of L. lactis C2 were compared with those of KB4 by use of a chemically defined medium, it was found that KB4 was unable to grow in the absence of aspartic acid. This aspartic acid requirement could also be met by aspartate-containing peptides. The addition of aspartic acid to milk restored the Fmc+ phenotype of KB4. KB4 was found to be defective in pyruvate carboxylase and thus was deficient in the ability to form oxaloacetate and hence aspartic acid from pyruvate and carbon dioxide. The results suggest that when lactococci are propagated in milk, aspartate derived from casein is unable to meet fully the nutritional demands of the lactococci, and they become dependent upon aspartate biosynthesis.

An origin of transfer (oriT) on the conjugative element pRS01 from Lactococcus lactis subsp. lactis ML3.

Previous analysis of the Tra1 region of the conjugative element pRS01 from Lactococcus lactis subsp. lactis ML3 suggested that an origin of transfer (oriT) was present. Deletion derivatives of this cloned Tra1 region were assayed for mobilization in the presence of the wild-type pRS01 element in trans. The pRS01 oriT was localized to a 446-nucleotide segment in the intergenic region between open reading frames ltrD and ltrE. Sequence analysis of this region revealed a cluster of direct and inverted repeat structures characteristic of oriT regions associated with other conjugative systems.

Homing of a group II intron from Lactococcus lactis subsp. lactis ML3.

Ll.ltrB is a functional group II intron located within a gene (ltrB) encoding a conjugative relaxase essential for transfer of the lactococcal element pRSO1. In this work, the Ll.ltrB intron was shown to be an independent mobile element capable of inserting into an intronless allele of the ltrB gene. Ll.ltrB was not observed to insert into a deletion derivative of the ltrB gene in which the intron splice site was removed. In contrast, a second vector containing a 271-nucleotide segment of ltrB spanning the Ll.ltrB splice site was shown to be a proficient recipient of intron insertion. Efficient homing was observed in the absence of a functional host homologous recombination system. This work demonstrates that the Ll.ltrB intron is a novel site-specific mobile element in lactococci and that group II intron self-transfer is a mechanism for intron dissemination among bacteria.

Characterization of the Highly Autolytic Lactococcus lactis subsp. cremoris Strains CO and 2250.

Two highly autolytic Lactococcus lactis subsp. cremoris strains (CO and 2250) were selected and analyzed for their autolytic properties. Both strains showed maximum lysis when grown in M17 broth containing a limiting concentration of glucose (0.4 to 0.5%) as the carbohydrate source. Lysis did not vary greatly with pH or temperature but was reduced when strains were grown on lactose or galactose. Growth in M17 containing excess glucose (1%) prevented autolysis, although rapid lysis of L. lactis subsp. cremoris CO did occur in the presence of 1% glucose if sodium fluoride (an inhibitor of glycolysis) was added to the medium. Maximum cell lysis in a buffer system was observed early in the stationary phase, and for CO, two pH optima were observed for log-phase and stationary-phase cells (6.5 and 8.5, respectively). Autolysins were extracted from the cell wall fraction of each strain by using either 4% sodium dodecyl sulfate (SDS), 6 M guanidine hydrochloride, or 4 M lithium chloride, and their activities were analyzed by renaturing SDS-polyacrylamide gel electrophoresis on gels containing Micrococcus luteus or L. lactis subsp. cremoris CO cells as the substrate. More than one lytic band was observed on each substrate, with the major band having an apparent molecular mass of 48 kDa for CO. Each lytic band was present throughout growth and lysis. These results suggest that at least two different autolytic enzymes are present in the autolytic L. lactis subsp. cremoris strains. The presence of the lactococcal cell wall hydrolase gene, acmA (G. Buist, J. Kok, K. J. Leenhouts, M. Dabrowska, G. Venema, and A. J. Haandrikman, J. Bacteriol. 177:1554-1563, 1995), in strains 2250 and CO was confirmed by Southern hybridization. Analysis of an acmA deletion mutant of 2250 confirmed that the gene was involved in cell separation and had a role in cell lysis.

Splicing of a group II intron involved in the conjugative transfer of pRS01 in lactococci.

Analysis of a region involved in the conjugative transfer of the lactococcal conjugative element pRS01 has revealed a bacteria] group II intron. Splicing of this lactococcal intron (designated Ll.ltrB) in vivo resulted in the ligation of two exon messages (ltrBE1 and ltrBE2) which encoded a putative conjugative relaxase essential for the transfer of pRS01. Like many group II introns, the Ll.ltrB intron possessed an open reading frame (ltrA) with homology to reverse transcriptases. Remarkably, sequence analysis of ltrA suggested a greater similarity to open reading frames encoded by eukaryotic mitochondrial group II introns than to those identified to date from other bacteria. Several insertional mutations within ltrA resulted in plasmids exhibiting a conjugative transfer-deficient phenotype. These results provide the first direct evidence for splicing of a prokaryotic group II intron in vivo and suggest that conjugative transfer is a mechanism for group II intron dissemination in bacteria.

Loss of plasmid-mediated oligopeptide transport system in lactococci: another reason for slow milk coagulation.

Fast milk-coagulating (Fmc+) strains of lactococci are known to segregate slow milk-coagulating (Fmc-) variants, which has been attributed to loss of proteinase (Prt) activity encoded by plasmid DNA. It was found that the Fmc- phenotype could also be due to loss of a plasmid encoding an oligopeptide permease (Opp) system. In Lactococcus lactis subsp. lactis (L. lactis) C2O, lactose metabolism (Lac) and Prt were linked to pJK550 and the Opp system to pJK430. In Lactococcus lactis subsp. cremoris SK11, known to possess Prt on a 78-kb plasmid, DNA sequence analysis of a 7.4-kb region from the Lac plasmid, pSK11L, revealed that it possessed the Opp system. The Lac plasmid in L. lactis C2 encoded both the Prt and Opp systems. Fmc- derivatives of L. lactis C2 were missing the prt genes and had Opp integrated into the chromosome, possibly due to transposition events. Growth studies showed the Opp systems were functional and, in combination with Prt, produced the Fmc+ phenotype.

Lactococcin A overexpression in a Lactococcus lactis subsp. lactis transformant containing a Tn5 insertion in the lcnD gene.

Lactococcin A production in lactococci has recently been linked to a signal-sequence-independent secretory system consisting of a four-gene cluster. Lactococcus lactis subsp. lactis LLM23L-A1 has been obtained after Tn5 mutagenesis of pLLM23, a plasmid containing the gene cluster responsible for lactococcin A production. In contrast to other Tn5-generated mutants, strain LLM23L-Al exhibited a 12-fold increase in lactococcin A production. Overproduction of lactococcin A was not linked to an increased pLLM23 copy number. Restriction-enzyme analysis indicated the site of Tn5 insertion to be at the 3' end of lcnD, and upstream of the lcnA structural gene. From DNA sequencing, the Tn5 insertion was located -79 bp upstream of the transcription start site of the lcnA and lciA genes, eliminating eight amino acids from the C-terminal end of lactococcin D. Northern blots revealed overproduction of a 500-base transcript in strain LLM23L-A1, which corresponded to that predicted from the positions of the lactococcin A operon transcriptional start site and the termination structures. This result suggests that the overproduction of lactococcin A in strain LLM23L-A1 is at the transcriptional level and provides further impetus for elucidating the complete regulatory mechanism for lactococcin A expression.

Novel insertion sequence-like element IS982 in lactococci.

A novel insertion sequence-like (IS) element, designated IS982, was found on the lactose plasmid, pSK11L, from Lactococcus lactis subsp. cremoris SK11 and was located between the origin of replication and the oligopeptide transport gene cluster. The 1003-base pair (bp) IS982 was flanked by 18-bp perfect inverted repeats. IS982 contained an open reading frame encoding a putative transposase of 296 amino acids. An almost identical IS-like element (99% DNA sequence identity) was cloned and partially sequenced from the chromosome of Lactococcus lactis subsp. cremoris Wg2 with 17-bp perfect inverted repeats. Southern analysis indicated that in 12 lactococcal strains examined, IS982 was present with copy numbers ranging from 1 to at least 20. IS982 displayed sequence homology to the putative IS element RSBst-alpha from Bacillus stearothermophilus CU21, IS982 and RSBst-alpha were not related to other known insertion sequences and may represent a new family of IS elements.

Cloning and characterization of the abortive infection genetic determinant abiD isolated from pBF61 of Lactococcus lactis subsp. lactis KR5.

A 6.3-kb fragment from pBF61 in Lactococcus lactis subsp. lactis KR5 was cloned and found to confer an abortive phage infection (Abi+) phenotype exhibiting a reduction in efficiency of plating and plaque size for small isometric- and prolate-headed bacteriophages sk1 and c2, respectively, and to produce a 10-fold decrease in c2 phage burst size. Phage adsorption was not significantly reduced. An open reading frame of 1,098 bp was sequenced and designated abiD. Tn5 mutagenesis confirmed that abiD was required for the Abi+ phenotype.

Genetic analysis of regions of the Lactococcus lactis subsp. lactis plasmid pRS01 involved in conjugative transfer.

The genes responsible for conjugative transfer of the 48.4-kb Lactococcus lactis subsp. lactis ML3 plasmid pRS01 were localized by insertional mutagenesis. Integration of the IS946-containing plasmid pTRK28 into pRS01 generated a pool of stable cointegrates, including a number of plasmids altered in conjugative proficiency. Mapping of pTRK28 insertions and phenotypic analysis of cointegrate plasmids identified four distinct regions (Tra1, Tra2, Tra3, and Tra4) involved in pRS01 conjugative transfer. Tra3 corresponds closely to a region previously identified (D. G. Anderson and L. L. McKay, J. Bacteriol. 158:954-962, 1984). Another region (Tra4) was localized within an inversion sequence shown to correlate with a cell aggregation phenotype. Tra1 and Tra2, two previously unidentified regions, were located at a distance of 9 kb from Tra3. When provided in trans, a cloned portion of the Tra3 region complemented Tra3 mutants.

Copy number and location of insertion sequences ISS1 and IS981 in lactococci and several other lactic acid bacteria.

Genomic DNA from 49 lactococcal strains was screened by Southern hybridization for the presence and relative copy number of lactococcal insertion sequence ISS1: ISS1 was found in 47 of 49 strains giving 1 to 20 hybridizing bands per strain. Southern hybridizations of undigested plasmid DNA from 17 lactococcal strains probed with ISS1 and IS981 showed that ISS1 was present on plasmids in all 17 strains, whereas IS981 was present on plasmids in 14 of the 17 strains. Both insertion sequences were present primarily on larger plasmids (> 25 kb), and some plasmids contained copies of both insertion sequences. When probed with ISS1, Southern hybridizations of DNA isolated from Lactococcus lactis ssp. lactis ML3 frozen stock culture and from isolated colonies showed that the stock culture consisted of a mixture of cells having different ISS1-hybridizing bands, indicating that stock cultures may contain cells with varying locations of ISS1 sequences. The number of copies and their widespread distribution among lactococcal strains establish that insertion sequences will contribute significantly to genotypic and phenotypic events that may affect the industrial performance and stability of lactococcal strains.