A soluble autolysin has been isolated from cell walls of exponential (log) phase cultures of Streptococcus faecalis ATCC 9790 by means of gel filtration of trypsin-activated wall lysates. Trypsin and other proteolytic enzymes speed wall autolysis apparently by "activating" the autolysin. The autolysin seems to be closely associated with the wall as virtually complete wall dissolution is a prerequisite for obtaining the enzyme in solution. While the soluble autolysin can act on walls of S. faecalis and Micrococcus lysodeikticus, it is virtually inactive on walls from several other bacterial species, Borohydride reduction indicates P revious studies (Shockman, 1963b(Shockman, , 1965Shockman et al., 1958 Shockman et al., , 1961Toennies and Shockman, 1958;Conover et al., 1966) have demonstrated that cells from exponential phase (log) cultures of Streptococcus faecalis 9790 will autolyze rapidly when placed under environmental conditions which prevent the continued synthesis of cell wall peptidoglycan (mucopeptide, glycopeptide, or murein). Inhibition of peptidoglycan synthesis (resulting in autolysis) can be caused either by the addition of a specific inhibitor, such as penicillin or cycloserine, or by deprivation of a nutritionally essential precursor of wall peptidoglycan, such as L-lysine, D-alanine, aspartic and glutamic acids, or glucose.The potential for cell autolysis reached a maximum toward the end of the exponential growth phase and thereafter the cells became resistant to autolysis (Shockman, 1965). Stationary-phase, valine-deprived (Val) or threonine-deprived (Thr) cultures resist autolysis (Shockman, 1965 ; Shockman et al., 1961).Two observations implicated an enzymatic attack on the rigid and protective cell wall as the initial step in cell autolysis. First, when an osmotically protective agent, such as 0.5 M sucrose, was provided, wallfree spherical, osmotically fragile protoplasts could be obtained from log cells, but not from stationaryphase Thr cells in the absence of an exogenous lytic ~~~

Two novel procedures have been used to regulate, in vivo, the formation of phosphoenolpyruvate (PEP) from glycolysis in Streptococcus lactis ML3. In the

In the absence of an exogenous energy source, galactose-grown cells of Streptococcus lactis Mb3 rapidly accumulated thiomethyl-,8-galactopyranoside (TMG) and 2-deoxyglucose to intracellular concentrations of 40 to 50 mM. Starved cells maintained the capacity for TMG uptake for many hours, and accumulation of the f8-galactoside was insensitive to proton-conducting ionophores (tetrachlorosalicylanilide and carbonylcyanide-m-chlorophenyl hydrazone) and sulfydryl group reagents including iodoacetate and N-ethylmaleimide. Fluorimetric analysis of glycolytic intermediates in extracts prepared from starved cells revealed (a) high intracellular levels of phosphoenolpyruvate (13 mM; PEP) and 2-phosphoglycerate (-39 mM; 2-PG), but (b) an absence of other metabolites including glucose 6-phosphate, fructose 6-phosphate, fructose 1,6diphosphate, and triosephosphates. The following criteria showed PEP (and 2-PG) to be the endogenous energy source for TMG accumulation by the phosphotransferase system: (i) the intracellular concentrations of PEP and 2-PG decreased with concomitant uptake of TMG, and a close correlation was observed between maximum accumulation of the B-galactoside and the total available concentration of the two intermediates; (ii) TMG accumulated as an anionic derivative, which after extraction and incubation with alkaline phosphatase (EC 3.1.3.1) formed the original analogue; (iii) fluoride inhibition of 2-phospho-Dglycerate hydrolyase (EC 4.2.1.11) prevented the conversion of 2-PG to PEP, and uptake of TMG by the starved cells was reduced by 80%; and (iv) the stoichiometric ratio [TMG] accumulated/[PEP] consumed was almost unity (0.93). In cells metabolizing glucose, all intermediates listed in (a) and (b) were found. Upon exhaustion of glucose from the medium, the metabolites in (b) were not longer detectable, while the intracellular concentrations of PEP and 2-PG increased to the levels previously observed in starved cells. The glycolytic intermediates in (b) are all in vitro heterotropic effectors of pyruvate kinase (adenosine 5'triphosphate:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from S. lactis ML3. It is suggested that the capacity of starved cells to maintain high intracellular concentrations of PEP and 2-PG is a consequence of decreased in vivo activity of this key regulatory enzyme of glycolysis. Streptococcus lactis and S. faecalis possess certain physiological characteristics (10, 11, 14) that have proved extremely useful in the study of energy coupling to solute accumulation by microorganisms (9, 10, 34). For example, under normal growth conditions these organisms lack a functional cytochrome system and so are unable to produce adenosine 5'-triphosphate (ATP) via oxidative phosphorylation. S. lactis and S. faecalis usually rely upon glycolysis or arginine catabolism for the generation of the compounds that serve directly (phosphoenolpyruvate, PEP) or indirectly (ATP) as energy

Starved cells of Streptococcus lactis ML3 (grown previously on galactose, lactose, or maltose) accumulated methyl-,8-D-thiogalactopyranoside (TMG) by the lactose:phosphotransferase system. More than 98% of accumulated sugar was present as a phosphorylated derivative, TMG-6-phosphate (TMG-6P). When a phosphotransferase system sugar (glucose, mannose, 2-deoxyglucose, or lactose) was added to the medium simultaneously with TMG, the ,-galactoside was excluded from the cells. Galactose enhanced the accumulation of TMG-6P. Glucose, mannose, lactose, or maltose plus arginine, when added to a suspension of TMG-6P-loaded cells of S. lactis ML3, elicited rapid expulsion of intracellular solute. The material recovered in the medium was exclusively free TMG. Expulsion of galactoside required both entry and metabolism of an appropriate sugar, and intracellular dephosphorylation of TMG-6P preceded efflux of TMG. The rate of dephosphorylation of TMG-6P by perrneabilized cells was increased twoto threefold by adenosine 5'-triphosphate but was strongly inhibited by fluoride. S. lactis ML3 (DGr) was derived from S. lactis ML3 by positive selection for resistance to 2-deoxy-D-glucose and was defective in the enzyme II"M component of the glucose:phosphotransferase system. Neither glucose nor mannose excluded TMG from cells of S. lactis ML3 (DGr), and these two sugars failed to elicit TMG expulsion from preloaded cells of the mutant strain. Accumulation of TMG-6P by S. lactis ML. can be regulated by two independent mechanisms whose activities promote exclusion or expulsion of galactoside from the cell. The accumulation ofglucose, lactose, and nonmetabolizable analogs 2-deoxy-D-glucose (2-DG) and methyl-,8-D-thiogalactopyranoside (TMG) I (2DG-6P) EIIac/EllAc HPr + lactose 6-phosphate (TMG-6P) (where PEP is phosphoenolpyruvate; EI, EHm', components of the PTS. In a previous study (40) EIIIAC, and EIIJC are enzymes I, [HM, HI',C and it was found that glucose, 2-DG, and lactose

Cell walls from exponential-phase cultures of Streptococcus faecalis ATCC 9790 autolyzed in dilute buffers. Walls were isolated from cultures grown in the presence of '4C-lysine for about 10 generations and then on 12C-lysine for 0.1 to 0.8 of a generation (prelabeled). These walls released 14C to the soluble fraction more slowly than they lost turbidity during the initial stages of autolysis. Walls isolated from cultures grown in the presence of '4C-lysine for only the last 0.1 to 0.4 of a generation (postlabeled) released 14C to the supernatant fluid more rapidly than they lost turbidity. Autolysin in both pre-and postlabeled walls was inactivated, and such walls were then incubated in the presence of unlabeled walls containing active autolysin. The inactivated walls lost their "IC label only very slowly until autolysis