The marine bacterium Pseudoalteromonas sp. strain A28 was able to kill the diatom Skeletonema costatum strain NIES-324. The culture supernatant of strain A28 showed potent algicidal activity when it was applied to a paper disk placed on a lawn of S. costatum NIES-324. The condensed supernatant, which was prepared by subjecting the A28 culture supernatant to ultrafiltration with a 10,000-M w -cutoff membrane, showed algicidal activity, suggesting that strain A28 produced extracellular substances capable of killing S. costatum cells. The condensed supernatant was then found to have protease and DNase activities. Two Pseudoalteromonas mutants lacking algicidal activity, designated NH1 and NH2, were selected after N-methyl-N-nitrosoguanidine mutagenesis. The culture supernatants of NH1 and NH2 showed less than 15% of the protease activity detected with the parental strain, A28. The protease was purified to homogeneity from A28 culture supernatants by using ion-exchange chromatography followed by preparative gel electrophoresis. Paper-disk assays revealed that the purified protease had potent algicidal activity. The purified protease had a molecular mass for 50 kDa, and the N-terminal amino acid sequence was determined to be Ala-Thr-Pro-Asn-Asp-Pro. The optimum pH and temperature of the protease were found to be 8.8 and 30°C, respectively, by using succinyl-Ala-Ala-ProPhe-p-nitroanilide as a substrate. The protease activity was strongly inhibited by phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate, antipain, chymostatin, and leupeptin. No significant inhibition was detected with EDTA, EGTA, phenanthroline or tetraethylenepentamine. These results suggest that Pseudoalteromonas sp. strain A28 produced an extracellular serine protease which was responsible for the algicidal activity of this marine bacterium.There have recently been discussions concerning the roles of marine bacteria in algal bloom dynamics (7,15,22). Marine bacteria may both promote and regulate algal blooms (6, 9). The fact that marine bacteria selectively promote bloom formation by algal species has recently been reported (10). On the basis of laboratory experiments, it has also been reported that some bacteria are able to inhibit the growth of red-tide algae (12). In general, bacteria that inhibit algal growth are effective through direct or indirect attack (2, 17). For example, the gliding bacterium Cytophaga sp. strain J18/M01 effectively kills diatoms and raphidophytes when it is added to algal cultures but not when filtrate alone is added (direct attack) (12). Indirect attacks are thought to be chemically mediated (17). Recent studies have demonstrated the presence of bacteria that lyse algal cells by producing extracellular substances (2, 8). Alga-lytic bacteria have also been found in coastal environments where harmful algal blooms often occur (2, 8, 12-14, 17, 19, 23). It is therefore possible that bacteria having algicidal effects are involved in the termination and decomposition of algal blooms. However, virtually nothing is known...

ATP-dependent glucokinase is suggested to have evolved from a hypothetical polyphosphate (polyP)-dependent glucokinase (polyP-GK) via a bifunctional polyP/ATP glucokinase (polyP/ATP-GK). Here we showed that polyP-GK is present in a polyP-accumulating bacterium, Microlunatus phosphovorus. The polyP-GK produced glucose-6-P i from glucose and polyP, but it could not phosphorylate glucose with ATP. The polyP-GK was most closely related to the polyP/ATP-GK of Mycobacterium tuberculosis.Inorganic polyphosphate (polyP) is a linear polymer of tens or hundreds of orthophosphate (P i ) residues linked in the same manner as the two high-energy phosphoanhydride bonds in ATP (4, 5). PolyP is readily formed by dehydration of P i and found in abundance in volcanic condensates and deep-oceanic steam vents (14). Hence, ancient organisms may have utilized polyP instead of ATP in their metabolic reactions (4,9,14).Glucokinases that use ATP as the sole phosphoryl donor to catalyze the phosphorylation of glucose (ATP-GKs) have been present in all contemporary organisms examined (2). Bifunctional glucokinase (polyP/ATP-GK), which utilizes polyP or ATP as the phosphoryl donor to phosphorylate glucose, was found first in Mycobacterium phlei (10) and then in many other bacteria, including Corynebacterium diphtheriae (11), Mycobacterium tuberculosis (12), and Propionibacterium shermanii (13). The polyP/ATP-GK of M. tuberculosis also utilizes GTP, UTP, and CTP as phosphoryl donors (12). PolyP is recognized as one of the earliest biopolymers and is most likely a prominent precursor in prebiotic evolution (4). Thus, it has been hypothesized that glucose phosphorylation was originally mediated by polyP and that when ATP became available in the environment, a transition was made by the GKs to utilize the latter phosphoryl donor (9). However, nobody has discovered the strictly polyP-dependent glucokinase that utilizes polyP as the sole phosphoryl donor (polyP-GK).Microlunatus phosphovorus strain NM-1 is a gram-positive, coccus-shaped, non-spore-forming bacterium (7). Strain NM-1, which was originally isolated with an enhanced biological phosphorus removal process, accumulates large amounts of polyP (a maximum of approximately 48% of its dry weight as phosphate [P i ]) in a glucose medium (7). We discovered the existence of polyP-GK in M. phosphovorus strain NM-1.M. phosphovorus strain NM-1 was grown for 2 days under aerobic conditions at 27°C in the glucose medium (7). The cells harvested at the mid-logarithmic phase of growth were disrupted by using Bead-beater (Biospec), and the polyP-GK was precipitated by adding 70% ammonium sulfate. The polyP-GK was further purified by using a DEAE-cellulose (Whatman), a phenyl-Sepharose (Amersham Biosciences), a PE hydrophobic (Poros), and two anion exchange (HQ [Poros] and miniQ [Amersham Biosciences]) columns. The polyP-GK was purified 253-fold (Fig. 1A), and the recovery of polyP-GK activity was 6.7%. The polyP-GK migrated as a 32-kDa protein on sodium dodecyl sulfate (SDS)-polyacrylamide gels, while...

No abstract