Keywords : phosphotransferase system, inducer expulsion, carbon and energy metabolism OverviewOver the past quarter of a century, tremendous effort has allowed elucidation of crucial aspects of the mechanisms of catabolite repression and cytoplasmic inducer control in Escherichia coli and other Gram-negative enteric bacteria (Botsford & Harman, 1992;Postma et al., 1993; Saier, 1989Saier et al., 1996). The extent of this effort reflected in part the belief that 'What is true for E. coli is also true for elephants' (J. Monod), and that the mechanism observed for E. coli would therefore prove to be universal. The observation that catabolite repression and inducer exclusion (Magasanik, 1970) are universal phenomena, documented in phylogenetically distant bacteria as well as in eukaryotes, seemed to substantiate this suggestion . Early investigations consequently focused on E. coli for a detailed understanding of the mechanisms involved. More recently, several research groups have begun to examine the mechanisms controlling carbohydrate catabolism in bacteria other than E. coli. In most cases, clear mechanistic concepts have not yet crystallized. However, in one group of prokaryotes, the low-GC Gram-positive bacteria, crucial aspects of the underlying mechanism are emerging. The proposed mechanism involves the proteins of the phosphoenolpyruvate (PEP)-dependent sugar transporting phosphotransferase system (PTS) as in E. coli, but the proteins that are directly involved in regulation and the mechanisms responsible for this control are completely different. This review provides a synopsis of recent advances concerned with the details of this process. The bacterial phosphotransferase system catalyses the concomitant uptake and phosphorylation (group translocation) of its sugar substrates (PTS sugars) via the PTS phosphoryl transfer chain as follows : PEP + Enzyme I + HPr + Enzyme IIASUg"' + Enzyme IIBCSUgar + sugar-P Virtually all low-GC Gram-positive bacteria that have been examined, including species of Bacilhs, Stapbylococczis, Streptococczrs, Lactococcus, Lactobacillzrs, Enterococczrs, Mycoplasma, Acboleplasma, Clostridizrm and Listeria, possess the enzymes of the PTS. In select Gram-positive bacteria, glucose has been shown to repress synthesis of both PTS and non-PTS carbohydrate catabolic enzymes ; it also inhibits the uptake of both PTS and non-PTS sugars (inducer exclusion) while stimulating dephosphorylation of intracellular sugar-Ps and/or efflux of the free sugars (inducer expulsion) (Fig. 1). Substantial evidence supports the contention that a metabolite-activated ATP-dependent protein kinase phosphorylates a seryl residue in HPr to regulate enzyme synthesis, inducer exclusion and inducer expulsion. A single allosteric regulatory mechanism acting on different target proteins is probably involved (Deutscher e t

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In the Gram-positive bacterium, Lactococcus lactis, nonmetabolizable cytoplasmic sugar phosphates, accumulated by the phosphoenolpyruvate:sugar phosphotransferase system, are rapidly dephosphorylated and expelled from the cell upon addition of glucose (inducer expulsion). Our recent studies have established that a metabolite-activated, ATP-dependent protein kinase that phosphorylates serine-46 in HPr of the phosphoenolpyruvate:sugar phosphotransferase system activates a sugar phosphate phosphatase, thus initiating the inducer expulsion process. A membrane-associated, HPr(Ser(P))-dependent phosphatase has been identified, solubilized from the membrane, separated from other cellular phosphatases, and purified to near homogeneity. It exhibits a low subunit molecular mass (10 kDa) and behaves on gel filtration columns like a monomeric enzyme. It has broad substrate specificity, optimal activity between pH 7.0 and 8.0, is dependent on a divalent cation for activity, and is not inhibited by fluoride. It is stimulated more than 10-fold by HPr(Ser(P)) or a mutant derivative of HPr, S46D HPr, in which the regulatory serine is changed to aspartate, which bears a permanently negative charge as does phosphate. Stimulation is due both to an increase in the maximal velocity (Vmax) and a decrease in the Michaelis-Menten kinetic constant (Km) for sugar phosphate. The enzyme exhibits a Ka for S46D HPr of 15 microM. Although the enzyme is thermally stable, activation by HPr(Ser(P)) is heat sensitive.

Heterofermentative gram-positive bacteria are believed to metabolize sugars exclusively via the pentose phosphoketolase pathway following uptake via sugar:cation symport. Here we show that anaerobic growth of one such bacterium, Lactobacillus brevis, in the presence of fructose induces the synthesis of a phosphotransferase system and glycolytic enzymes that allow fructose to be metabolized via the Embden-Meyerhof pathway.Lactic acid bacteria are a group of gram-positive bacteria that characteristically lack cytochromes and are dependent on substrate level phosphorylation during sugar fermentation for energy (1). These bacteria are subdivided on the basis of their pathways and products of sugar fermentation. Homofermentative lactic acid bacteria use the Embden-Meyerhof pathway (EMP) to generate lactate as the sole product of fermentation, whereas heterofermentative lactic acid bacteria use the pentose phosphoketolase pathway (PKP) to produce a mixture of CO 2 , ethanol, acetate, and lactate (3, 11). While two molecules of phosphoenolpyruvate (PEP) are generated per sugar molecule metabolized via the EMP, only one PEP molecule results from sugar metabolism via the PKP. This fact has been used to rationalize the observation that bacteria that use the EMP usually have the PEP-dependent sugar transporting phosphotransferase system (PTS), while those which use the PKP do not. Indeed, the PTS has been reported to be widespread in homofermentative lactic acid bacteria but absent from heterofermentative bacteria (11,12). Lactobacillus brevis is one heterofermentative lactic acid bacterium which has been reported to lack PTSs specific for glucose, fructose, and lactose (10, 12). Glucose and lactose are known to be transported into L. brevis by sugar:H ϩ symport (10,20,21). Phosphofructokinase and fructose-1,6-bisphosphate aldolase have not been identified in this organism.The PTS catalyzes sugar uptake and phosphorylation via the following scheme: Sugar out ( PEP 3 I ϳ P 3 HPr ϳ P 3 IIA ϳ P 3 IIBC ϳ P 3 Sugar-P in n pyruvate I (enzyme I) and HPr (a heat-stable phosphocarrier protein of the PTS) are the general, energy coupling proteins of the system, while the IIA, IIB, and IIC proteins or protein domains are the constituents of the sugar-specific permeases (enzyme II complexes) that catalyze sugar transport and phosphorylation (6,15). While HPr has been detected in L. brevis, a search for additional components of the PTS gave negative results (9). It was therefore postulated that of the PTS proteins, this heterofermentative organism possesses only HPr and uses it exclusively for regulatory purposes. The use of HPr, the target of an ATP-dependent, metabolite-activated HPr(ser) kinase, to regulate both lactose:H ϩ symport and glucose:H ϩ symport in L. brevis has been extensively documented (9,10,13,(20)(21)(22)(23).We have previously reported that Listeria and Streptomyces species possess fructose-specific PTSs (8,17,18). While examining the properties of L. brevis, as described in Bergey's Manual of Systematic Bacteriolog...

Mangrove is a rich and underexploited ecosystem with great microbial diversity for discovery of novel and chemically diverse antimicrobial compounds. The goal of the study was to explore the pharmaceutical actinobacterial resources from mangrove soil and gain insight into the diversity and novelty of cultivable actinobacteria. Consequently, 10 mangrove soil samples were collected from Futian and Maoweihai of China, and the culture-dependent method was employed to obtain actinobacteria. A total of 539 cultivable actinobacteria were isolated and distributed in 39 genera affiliated to 18 families of 8 orders by comparison analysis of partial 16S rRNA gene sequences. The dominant genus was Streptomyces (16.0 %), followed by Microbacterium (14.5 %), Agromyces (14.3 %), and Rhodococcus (11.9 %). Other 35 rare actinobacterial genera accounted for minor proportions. Notably, 11 strains showed relatively low 16S rRNA gene sequence similarities (< 98.65 %) with validly described species. Based on genotypic analyses and phenotypic characteristics, 115 out of the 539 actinobacterial strains were chosen as representative strains to test their antibacterial activities against “ESKAPE” bacteria by agar well diffusion method and antibacterial mechanism by the double fluorescent protein reporter system. Fifty-four strains in 23 genera, including 2 potential new species, displayed antagonistic activity in antibacterial assay. Meanwhile, 5 strains in 3 genera exhibited inhibitory activity on protein biosynthesis due to ribosome stalling. These results demonstrate that cultivable actinobacteria from mangrove soil are potentially rich sources for discovery of new antibacterial metabolites and new actinobacterial taxa.