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Item A study of the cell wall-associated proteinase of lactic streptococci : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in biochemistry at Massey University(Massey University, 1988) Ng, Kee HuatThe cell wall proteinase of Streptococcus lactis 4760 was released by incubation of milk grown cells in Ca++- free buffer. The effects of duration of incubation, pH and presence of Ca++ ions on the release of proteinase activity was investigated. The extent of leakage of intracellular enzymes during incubation was monitored by the appearance of lactate dehydrogenase activity in the incubation buffer. The proteinase released from the cells was partially purified by ion- exchange and gel permeation chromatography and then analysed for activity towards various milk- proteins. Only a single proteinase was evident from the purification. This enzyme was active towards - casein but showed no apparent cleavage of a 81- and K- caseins nor the whey proteins, a- lactalbumin and - lactoglobulin. The enzyme cleaved the - casein molecule within the C- terminal 49 residues, generating four main peptides containing residues 167- 175, 176- 182, 183- 193 and 194-209, and smaller amounts of peptides corresponding to the overlapping sequences 161- 166, 164- 169 and 166- 175. The four main peptides are identical to those generated by an S. lactis 763 proteinase described by Monnet et al. (1986) and by an S. cremoris HP proteinase recently described by Visser et al. (1987). No apparent specificity of enzyme action was evident. A preliminary study of the cell wall proteinase from S. cremoris SKl 1, a strain reported to produce a proteinase with a different specificity, suggested that the enzyme may hydrolyse the - casein molecule at the same sites as those cleaved by the S. lactis 4760 enzyme.Item Genomic characterization and evolutionary relationships among Bacteriophages in the dairy industry and applications to detect phage contamination : a thesis presented in fulfilment of the requirements for the degree of Master of Philosophy at Massey University, Auckland, New Zealand(Massey University, 2018) Rajan, PriankaThe prevalence of bacteriophages and their pernicious effects on the Lactococcus starter culture in the dairy industry has been an ongoing problem for several decades. The main purpose of this investigation was to understand the relationship, evolutionary history and the sources of the phages that have been isolated from the different fermentative units of Fonterra. We report the genomic comparison results of 15 phages in this study that were isolated on the host bacteria, Lactococcus lactis cremoris. These phages can be grouped in two clusters namely P335 and 936, commonly encountered tailed bacteriophages in the dairy industries. The majority of the phages belong to the P335 species with just one phage clustering with the 936 species. Although phages of the P335 group display a high level of synteny with one another, we report nine different types of P335 phages in this study. A prophage integrated in the host strain has been identified. The prophage and the phages show homology to the temperate P335 phage, R1T isolated in Netherlands in 1996. The genetic makeup of these phages is suggestive of their source and evolution from other prophages in strains that may have been co-cultured with the strain that was used in this study. Furthermore, we identified that horizontal gene transfer events and homologous recombination have played a role in the evolution of phages in our study. Phage annotation was carried out for representatives in both the clusters and forty eight to fifty seven ORFs have been identified in these phages. Our analyses indicate that majority of the genes are conserved across these phages. For further detection of phages, this project also suggests rapid tools like PCR that can be used to better understand the phage species and the type of phage infecting the starter culture. With the availability of whole genome sequences, we are hoping that the genome analysis will enlighten our knowledge on the current distribution of phages and their relationship with one another in the dairy industry.Item Purification, crystallization and cloning of tributyrin esterase from Lactococcus : a thesis presented in partial filfillment of the requirements for the degree of Master of Technology in Biotechnology in the Institute of Molecular Biosciences at Massey University, New Zealand(Massey University, 1998) Zheng, JiongTributyrin esterase is an enzyme that has been isolated and purified from lactococcal starter strain by research staff at the New Zealand Dairy Research Institute. It has been shown to play an important role in production and control of flavour development during cheese ripening, but little is known about its biochemical characteristics. New studies on tributyrin esterase have been initiated, with the aim of carrying out a three dimensional structure determination to completely understand the molecular basis and the nature of its in vivo activity. This thesis is divided into three main parts. In the first part, the purification of tributyrin esterase from a genetically modified strain Lc. lactis subsp. cremoris B1079 is described. The procedure investigated for optimization of the protocol and a partial study of factors affecting tributyrin esterase activity are described. In the second part, crystallization trials for tributyrin esterase are described. Several crystals were obtained, with the best ordered crystals being grown from 2.6M ammonium sulfate, these have been shown to diffract to 3.0 Å, and belong to the space group C222 with cell dimensions a=76Å, b=178Å c=179Å. In the third part, the lipase gene was ligated into 4.75kbp expression vector proEX, which contains a his-tag sequence upstream of the multiple cloning site. The ligation reaction mixture was transformed into competent E.coli DH5α cells. This should allow the expression of tributyrin esterase in E.coli and eventually provide a great yield of protein and make purification easier.Item Physiological changes associated with the appearance of slow variants in cultures of Streptococcus lactis : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University, New Zealand(Massey University, 1972) Skipper, Nigel ArmstrongStreptococcus lactis C10 Slow, a variant of the normal 'fast' strain of S.lactis C10, was only capable of rapid and extensive growth in skim milk if casein hydrolysates were added. It was postulated, therefore, that the slow variant is defectively proteolytic. A sensitive assay of proteolysis, based on the release of radio-activity from iodinated casein, was developed, checked for usefulness with known proteinases, then used to assay the streptococcal enzymes. Fractionation of the two strains, by either mechanical cell disruption and differential centrifugation or by cell-wall digestion with a muraminidase, established that most of the cell-bound proteinases of the parent strain were surface-bound. This activity was virtually absent in the slow variant. Partial characterization of the surface-proteinase(s) showed that maximum activity was exhibited at pH 6.0-6.8, in 0.05 M phosphate buffer, at 30-32°C. It was rapidly inactivated at 37°C, both when cell-bound and when free of the cells. Examination of a second pair of strains, S.lactis H1 Fast and S.lactis H1 Slow, indicated a difference in proteinase activity and localization similar to that found between the two S.lactis C10 strains. It was concluded, on the basis of both nutritional evidence and enzymatic analyses, that the slow variant of S.lactis C10 is limited in skim milk by the supply of amino acids and that this is due to a defective surface-bound proteolytic activity.Item Increasing the Lactococcus lactis biomass through aerobic growth : a thesis presented in partial fulfilment of the requirement for the degree of Master of Engineering in Biotechnology at Massey University, Palmerston North, New Zealand(Massey University, 2013) Giridhar, AravindStarter cultures for dairy fermentations are commonly made by anaerobic fermentation in New Zealand. Anaerobic fermentation involves glycolysis and it is a very inefficient pathway due to the formation of energy rich products such as lactic acid. This pathway only produces 2 moles of ATP per glycolysis and to conserve energy, the amount of biomass produced is less. Aerobic fermentation on the other hand can produce up to 36 moles of ATP per cycle, and the amount of biomass produced will be higher compared to anaerobic fermentation. Lactic acid bacteria do not possess a functional electron transport chain for aerobic respiration to be efficient. It requires the addition of heme, for the electron transport chain to work. The heme addition is a patented process. The aim of this study was to optimise the aerobic fermentation process for Lactococcus lactis for biomass production. An extensive literature search shows that there has been no study in optimising the heme concentration or using other alternatives for heme. Alternatives to heme, that are food grade, are an attractive option, as there is sourcing issues with heme in New Zealand. A series of shake flask trials were carried out to identify a possible heme replacement. The shake flask trials showed that ammonium ferric citrate is a possible alternative heme replacement. More shake flask trials were then evaluated to optimise the concentration of ammonium ferric citrate. Following that, 1-L fermenter trials were evaluated to optimise heme concentration and to compare the effect of heme and ammonium ferric citrate addition on biomass and activity of the harvested biomass following a freeze and thaw cycle. It was shown that 44 µg/mL ammonium ferric citrate resulted in the most biomass of the concentrations tested. For heme, the optimum concentration was 1 µg/mL. It was found that fermentations using heme resulted in more biomass after 5 h compared to using ammonium ferric citrate. But, cells grown by adding ammonium ferric citrate was equally as active.Item Metabolic engineering of Lactococcus lactis to enhance biopolymer bead production : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University, Palmerston North, New Zealand(Massey University, 2012) Hume, Lydia Shivonne-LeePolyhydroxyalkanoates (PHAs) are a group of biopolyesters that are synthesized by polyester synthases in a wide range of Gram-positive and Gram-negative bacteria, and are stored in bacterial cells as intracellular inclusions. Recently, these inclusions have been considered for biotechnological and biomedical applications as surface-functionalized micro-/nanobeads. The production of functionalized poly[(R)-3-hydroxybutyrate] (PHB; a biopolyester) beads in the food-grade host Lactococcus lactis has recently been established, however the levels of PHB production are low in comparison to levels produced by recombinant E. coli. In an attempt to improve PHB production in L. lactis, the metabolic flux of carbon from pyruvate was engineered to redirect the flux towards acetyl Co-A, one of the precursors for PHB. This involved knocking out two enzymes involved in conversion of acetyl Co-A to acetate and ethanol (acetaldehyde dehydrogenase and phosphate acetyltransferase, respectively). PHB production using a strain deficient in acetaldehyde dehydrogenase (adhE) was not assessed due to difficulties encountered in creating the knockout strain. This study showed the successful construction and phenotypic characterisation of a phosphate acetyltransferase (eutD) deficient strain of L. lactis. Production of acetate was substantially reduced in this mutant, and growth of the strain was improved when PHB production was established. However, rather than increasing, levels of PHB production by the eutD knockout were comparable to WT. Additionally, complementation of the mutant strain still needs to be achieved to confirm that the observed acetate-production phenotype is attributable to the lack of the eutD gene.Item Molecular analysis of the lactose metabolising genes from Lactococcus lactis : a thesis presented in partial fulfilment of the requirement for the degree of PhD in Biotechnology at Massey University, New Zealand(Massey University, 1993) Yunchalard, SirindaTwo lactococcal strains possessing different lactose metabolism systems were chosen for the molecular analysis of lactose metabolising genes from Lactococcus lactis. These were Lactococcus lactis ssp. cremoris strain H2 and Lactococcus lactis ssp. lactis strain ATCC 7962. An attempt was made to sequence a previously cloned 4.4 kb EcoRI fragment of pDI21 reported to encode D-tagatose 1,6-bisphosphate aldolase. A comparison of sequence data generated from this fragment with DNA sequences in the GenEMBL data base revealed that the clones provided for this study were not lactococcal DNA but were chromosomal DNA of E. coli which encoded genes involved in purine biosynthesis. This part of the research programme was therefore abandoned. The aim of the second part of this study programme was to clone and characterize the β-galactosidase gene from plasmid pDI3, an uncharacterized plasmid of Lactococcus lactis ssp. lactis strain ATCC 7962. This was of interest as most lactococcal bacterial strains metabolize lactose by way of the Lac-PEP:PTS system and possess high phospho-β-galactosidase activity, whereas strain 7962 metabolizes lactose by way of the lactose permease system and possesses high β-galactosidase activity and low phospho-β-galactosidase activity. Previous plasmid curing experiments indicated that the β-galactosidase activity of strain 7962 is associated with pDI3. DNA hybridization work between pDI3 and a previously cloned DNA fragment containing the β-galactosidase gene of another Gram-positive genus, Clostridium acetobutylicum (cbgA) showed that pDI3 contains DNA sequence that is to some extent homologous to β-galactosidase sequence. Initial experiments were carried out to confirm the involvement of pDI3 on strain 7962's β-galactosidase activity. Strain ATCC 7962 wildtype containing four plasmids and the strain cured of three other plasmids (i.e. derivative strain of 7962 containing only pDI3) exhibited a Lac+ phenotype, while the strain cured of all four plasmids exhibited a Lac- phenotype. As part of the mapping strategy and the attempt to clone the β-galactosidase gene from strain 7962, various fragments of pDI3 were cloned. Hybridization experiments using the cloned pDI3 fragments as DNA probes were also carried out to confirm the arrangement of pDI3 fragments. A physical map of pDI3 was constructed using the restriction enzymes BamHI, PstI, and SalI. The size of pDI3 was confirmed to be 70 kb and it may contain some small repeat sequences. Some EcoRI restriction sites contained in pDI3 were also determined. Several approaches were used to localize and identify the β-galactosidase gene on pDI3. Southern hybridizations were first carried out using the cbgA gene. The cbgA gene showed weak homology to a 4.3 kb EcoRI doublet from pDI3. Two redundant oligonucleotide probes were designed from the highly conserved domain of deduced amino acid sequences of the available β-galactosidase sequences from other closely related Gram-positive bacteria as well as some deduced amino acid sequences derived from β-galactosidase sequences of Gram-negative bacteria. The 4.3 kb EcoRI doublet and a 4.3 kb HindIII doublet exhibited weak homology to these probes. On the basis of these results one of the 4.3 kb EcoRI fragments was subsequently cloned and transformed into a Lac- E. coli strain, MC1022. The cloned 4.3 kb EcoRI fragment (the 4.3a fragment) was shown to cross hybridize to one of the oligonucleotide probes, but did not show β-galactosidase activity. The 4.3a kb EcoRI fragment was also cloned into pBR322 and transformed into another Lac- E. coli host (JM109) and no β-galactosidase activity was detected. Based on the pDI3 physical map constructed, the 4.3a kb EcoRI fragment was shown to overlap a 13.6 kb SalI fragment. This fragment was cloned into pBR322 and the plasmid designated pSY303, and this was transformed into E. coli JM109. Introduction of pSY303 into E. coli JM109 gave a Lac+ phenotype. Lac+ phenotypes were also found for other Lac- E. coli host strains including E. coli PB2959. It was found that pSY303 expressed β-galactosidase constitutively, however the addition of 0.1% (w/v) lactose into the medium gave a higher level of expression. An inducer for β-galactosidase, IPTG, was not required for expression. Glucose had no repression effect on β-galactosidase. Some instability of the Lac+ phenotype was observed. Transformants that were initially Lac+ manifested phenotypic segregation into Lac+ and Lac- colonies. Both were found to retain the intact pSY303 plasmid and there was no difference in pSY303 DNA isolated from representative Lac+ colonies and from representative Lac- colonies. Some stable Lac+ colonies were observed that became dark blue. Analysis of the plasmid from these colonies showed that pSY303 had undergone a deletion. The generation of these deletion derivatives may be a consequence of small repeat sequences. In conclusion, a physical map of pDI3 has been constructed and the β-galactosidase gene from pDI3 of Lactococcus lactis ssp. lactis strain ATCC 7962 was cloned and was found to express constitutively in E. coli, and at a higher level in the presence of lactose.Item The regulation of some glycolytic enzymes in streptococcus lactis : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, New Zealand(Massey University, 1975) Crow, Vaughan LeslieCertain aspects of the control of carbohydrate metabolism have been studied primarily in S. lactis C10. The kinetic and regulatory properties of two enzymes, lactate dehydrogenase and pyruvate kinase were investigated in some detail whereas a third enzyme, 6-phosphogluconate dehydrogenase, was subjected to a preliminary investigation only. A brief investigation was made of the in vivo concentrations of some metabolites in exponentially growing cells in batch culture. The S. lactis lactate dehydrogenase (LDH) was purified about 100 fold. The mobility pattern of the purified enzyme on polyacrylamide disc gel electrophoresis was a complex function of pH and ionic strength. From sodium dodecylsulphate-gel electrophoresis the LDH appeared to have a subunit molecular weight of 37,000. A tentative model indicating a pH dependent association/dissociation has been suggested on the basis of the gel results and heat stability studies. At acid and neutral pH values a tetrameric species is favoured. At alkaline pH values (pH 8.0) a dimeric species is favoured. The tetrameric protein is more stable to heat than the dimeric species. The purified LDH requires fructose-1 ,6-diphosphate (FDP) for catalytic activity at acid and neutral pHs. For pyruvate reduction, in the presence of FDP the pH optimum was 6.9 whereas in the absence of FDP only very low activity was found and the pH optimum was 8.0 to 8.2. The pH optimum for lactate oxidation in the presence of than the PDP activation of pyruvate reduction. The kinetics of lactate oxidation suggested that only the pyruvate reduction direction was significant in vivo. A significant finding was the effect of different buffers on the FDP activation of LDH. The concentration of FDP required for 50% maximal activity was 0.002 mM when determined in triethanolamine/HCl buffer, 0.2 mM in tris/maleate buffer and 4.4 mM in phosphate buffer; a 2,000 fold difference depending on the choice of the assay buffer. At the pH optimum (pH 6.9) there appeared to be at least two FDP binding sites which interact with each other in a co-operative manner. The choice of buffer was shown to affect other properties of LDH, such as the pH effect on FDP binding, the heat stability of the enzyme at 55°C, the binding of NADH and pyruvate and the effect of the inhibitor, oxamate. Stopped-flow analysis of the LDH showed that a lag period was present at pH 6.9. This lag period could be eliminated by pre-incubation with FDP. No such lag period was demonstrated at pH 8.2. It is suggested that this lag period is due to a conformational change in the tetrameric species induced by FDP. The properties of the S. lactis LDH, taking into account the buffer effects, have been discussed in terms of the carbohydrate metabolism and related to other FDP-activated streptococcal LDH's. A brief comparative study of the S. faecalis ATCC 8043 LDH was made. The two major findings were its insensitivity to phosphate inhibition and its activation by manganese ions. Pyruvate kinase was purified to near homogeneity as determined by polyacrylamide gel electrophoresis, with and without SDS. With SDS, a subunit molecular weight of 60,750 was determined. From equilibrium sedimentation studies the molecular weight of the native protein is 235,000. The enzyme is therefore a tetrameric protein. The kinetic properties of the pyruvate kinase were more complex than those of LDH, for as well as requiring FDP as an activator, the enzyme had an essential requirement for both a monovalent and divalent cation. FDP under most conditions bound to the enzyme in a co-operative manner. Phosphoenol-pyruvate (PEP), and to a lesser extent, ADP, showed co-operative binding to the enzyme only at unsaturating FDP concentrations. Both the monovalent and divalent cations showed co-operative binding to the enzyme in the presence of saturating FDP concentrations. The activation properties of the enzyme were considerably different when Mn++ was substituted for Mg++ as the divalent cation. Like LDH, the pyruvate kinase was also affected by the nature of the buffer components. Pyruvate kinase was inhibited by lower concentrations of phosphate than were required to inhibit LDH. In addition the pyruvate kinase activity was inhibited by high concentrations of Mg++ and ADP. The properties of the S. lactis pyruvate kinase have been discussed in relation to other pyruvate kinases and to carbohydrate metabolism in S. lactis. The S. lactis 6-phosphogluconate dehydrogenase (6-PGDH) did not appear to be inhibited by FDP, nor did the enzyme from S. faecalis ATCC 8043. This is contrary to published findings by other workers. Because of the preliminary nature of this investigation, further work is required on the S. lactis 6-PGDH to establish whether or not its activity is regulated by FDP. The in vivo concentration of several metabolites were determined in exponentially growing cells and related to the in vitro kinetic properties of the two enzymes, LDH and pyruvate kinase. The metabolites studied were; FDP, PEP, triose phosphates, ADP, ATP, glucose-6-phosphate and pyruvate. The in vivo FDP concentration was at a sufficiently high level (12.7 to 14.9 mM) to fully activate the two enzymes as indicated by in vitro determinations under a number of different assay conditions. The in vivo studies have suggested further in vitro kinetic studies which may be useful to investigate to gain a fuller understanding of the regulation of carbohydrate metabolism in S. lactis.Item Metabolism of lactic acid bacteria : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, New Zealand(Massey University, 1968) Thomas, Terence DavidStreptococcus lactis organisms were grown in lactose-limited batch culture and the survival characteristics of washed organisms were examined at the growth temperature. Washed suspensions had high initial viabilities (>99%) which were maintained for varying periods depending on the presence of certain added materials in the buffer and the conditions of incubation. Added Mg2+ markedly prolonged survival, while high bacterial concentrations also extended survival times, probably because Mg2+ was excreted by the bacteria. Surviving organisms in some conditions showed prolonged division lags, especially in the absence of Mg2+. Addition of trace amounts of EDTA decreased the death rate by removing toxic cation impurities, while the buffer salt concentration had little effect on survival within wide limits. The optimum pH value for survival was near 7.0 and survival times increased considerably at lower temperatures. Agitation and aeration tended to decrease survival and the death rate was not influenced by the phase of growth at which the organisms were harvested from a lactose-limiting medium. Addition of casamino acids increased survival times markedly in the presence of Mg2+; arginine was almost as effective as the complete mixture of amino acids while other individual amino acids tested gave only slight increases in survival times. Fermentable carbohydrates accelerated death of starved organisms irrespective of the growth phase from which they were harvested and of the limiting nutrient; the accelerated death rate was reduced by addition of Mg2+. Glucose metabolism proceeded at a much faster rate than arginine metabolism, theoretically producing about 7.5 times as much ATP. This rapid generation of ATP may be responsible for the more rapid death rates with added carbohydrates. Arginine substantially reduced the lethal effect of adverse pH values and suppressed the leakage of free intracellular amino acids into the external medium. Survival studies were followed by an investigation of the changes which took place in starved or ganisms and their relation to survival. No polyglucose or poly-β-hydroxybutyrat was detected and starved organisms had a negligible respiration rate. Soluble protein was released from viable organisms into the suspending buffer and the intracellular free amino acid pool declined steadily with the components appearing in the suspending buffer; a net increase in the total amount of free amino acid indicated some protein hydrolysis. Chloramphenicol reduced the death rates in some environments, possibly by suppressing protein degradation. RNA was hydrolysed with the release of u.v.-absorbing bases and ribose from the organisms. Conditions which promoted rapid RNA breakdown also produced rapid death rates and long cell division lags in surviving organisms. There was no appreciable degradation of carbohydrate or DNA. After 28 hr. starvation in buffer containing Mg2+, the bacterial dry wt. decreased by 26%; loss of RNA, protein and free amino acids accounted for 10.3%, 7.3% and 2.7% of the total bacterial mass loss. The products of polymer hydrolysis appeared to be released in an undegraded form into the external buffer and there was no appreciable formation of lactate, ammonia or volatile fatty acids possibly indicating the absence of any important endogenous energy sources. Protein synthesis, determined by the incorporation of valine-14C into TCA-insoluble material, was barely detectable when organisms were starved in buffer containing Mg2+. Addition of an energy source allowed limited protein synthesis while glucose produced a much higher rate of valine-14C uptake and incorporation than arginine. Although arginine prolonged survival this was not due to the limited protein synthesis which took place. The survival capacity of starved organisms could be correlated with the ability to synthesize protein which in turn may be correlated with RNA stability. A new method was developed for the assay of glycolytic activity in microorganisms. Organisms were incubated with glucose-U-14C and samples removed at intervals. Samples were chromatographed on DEAE-cellulose paper strips in deionized water which separated the radioactive anionic products of glycolysis (lactate, acetate and formate) from the unfermented glucose. The activity of the two fractions was then determined by liquid scintillation counting. The glycolytic activity of starved organisms declined steadily and was not correlated with survival. Phospholipid was broken down on prolonged starvation and the permeability properties of the organism were gradually lost. Addition of spermine gave enhanced survival and suppressed the release of u.v.-absorbing material. Lactic dehydrogenase and DNA were released as the death rate increased in buffer containing Mg2+ and eventually, well after death, cell lysis occurred. Electron micrographs indicated that addition of amino acids maintained cell structures for a much longer period and in this system cell lysis occurred as the death rate increased. It was concluded that the death rate of starved S. lactis organisms in phosphate buffer was partly dependent on the presence of Mg2+, which probably acted by promoting polymer stability, particularly that of RNA. In this environment, a suitable exogenous energy source further enhanced survival which may ultimately be a function of cell wall and membrane stability.