An analysis of the biochemical basis for the lack of phosphoenolpyruvate:glycose phosphotransferase ac- tivity in heterofermentative lactobacili was carried out. Ex- tracts of Lactobacillus brevis and Lactobacillus buchneri failed to reconstitute phosphotransferase activity of extracts of Staphylococcus aureus mutants impaired in the phosphotrans- ferase system due to the absence of enzyme I, enzyme flLac, or enzyme HILac activity, suggesting that these lactobacilli lack those phosphotransferase system components. In contrast, complementation tests with an extract of a S. aureus mutant deficient in heat-stable protein (HPr) indicated the presence of HPr activity in heterofermentative lactobacilli. The HPr of L. brevis was purified and shown to have properties similar to those of a typical HPr. In addition, L. brevis possesses an ATP-dependent protein kinase that phosphorylates a serine residue of the endogenous HPr as well as other HPrs of Gram-positive origin. The kinase activity is markedly stimu- lated by phosphorylated compounds related to sugar metabo- lism and is negatively modulated by orthophosphate, pyro- phosphate, or arsenate and by a low molecular weight endog- enous factor. In keeping with the idea of a regulatory role for the phosphorylation of HPr in lactobacilli, a HPr(Ser(P)) phosphatase activity in L. brevis was also demonstrated. On the basis of the finding of HPr and a system for its reversible covalent modification in an organism devoid of a functional phosphotransferase system we propose that, in lactobacilli, HPr has a role in the regulation of pathways other than the phosphotransferase system.

Since representatives of most classes of permeases have been characterized more extensively in gram-negative bacteria or eukaryotes than in gram-positive bacteria, this chapter provides comparative information with a focus on the unique features of specific well-characterized transport systems in representative gram-positive bacteria. These transport systems are energized by ATP hydrolysis, consumption of chemiosmotic energy in the form of ion gradients and membrane potentials, or phosphoryl transfer from PEP to the sugar substrate in the phosphotrans-ferase-catalyzed group translocation process. All three types of systems are found in gram-negative bacteria as well as in gram-positive bacteria, and representatives within all permease classes except the group translocating PTS permeasesare also found in eukaryotes. Although the transport proteins derived from grampositive bacteria are, in general, related to transport proteins of other bacteria and eukaryotes, the grampositive bacterial systems exhibit some unique properties. In some cases, these systems are characterized well enough that they provide information that clearly complements or contrasts with that obtained from the study of related transport systems of other organisms.

ABSTRACT Recent Escherichia coli genome sequencing efforts in the 76–81.5 min and 92.8–0.1 min regions have revealed three new operons or gene clusters concerned with sugar metabolism, which we designate sga (6 ORFs), sgb (4 ORFs), and sgc (6 ORFs). All of these operons encode proteins homologous to pentose-phosphate-4-epimerases ( sga and sgb ) or pentose-phosphate-3-epimerases ( sgc ), indicating that these operons are involved in the metabolism of pentoses or pentitols. sga and sgc (but not sgb ) encode also proteins of the bacterial phosphotransferase system (PTS), whereas sga and sgb encode three non-PTS proteins (one of which is the pentose-phosphate-4-epimerase homologue) that are homologous to each other. The two PTS protein homologues in the sga operon are members of (1) the family of mannitol-specific and fructose-specific IIA proteins and (2) the family of lactose-specific and cellobiose-specific IIB proteins. A IIC PTS homologue was not found in the sga operon, but a permease-like protein designated SgaT with 12 putative transmembrane helical segments may provide this transport function. Although the sgb operon lacks ORF homologous to PTS proteins, it contains a cryptic gene encoding L -xylulose kinase. The sgc gene cluster encodes two PTS proteins homologous to (1) the mannitol-specific and fructose-specific IIA proteins and (2) the galactitol IIC protein. It also encodes a transcriptional regulator of the DeoR family, a pentose-phosphatase-3-epimerase homologue, and an ORF homologous to a protein encoded in the recently described frv operon. Computer analyses of the DNA sequences and the encoded protein sequences are presented, and the potential roles of these operons in carbohydrate metabolism are discussed.

Results currently available clearly indicate that the metabolite-activated protein kinase-mediated phosphorylation of Ser-46 in HPr plays a key role in catabolite repression and the control of inducer levels in low-GC Gram-positive bacteria. This protein kinase is not found in enteric bacteria such as E. coli and Salmonella typhimurium where an entirely different PTS-mediated regulatory mechanism is responsible for catabolite repression and inducer concentration control. In Table 2 these two mechanistically dissimilar but functionally related processes are compared (Saier et al., 1995b). In Gram-negative enteric bacteria, an external sugar is sensed by the sugar-recognition constituent of an Enzyme II complex of the PTS (IIC), and a dephosphorylating signal is transmitted via the Enzyme IIB/HPr proteins to the central regulatory protein, IIAGlc. Targets regulated include (1) permeases specific for lactose, maltose, melibiose and raffinose, (2) catabolic enzymes such as glycerol kinase that generate cytoplasmic inducers, and (3) the cAMP biosynthetic enzyme, adenylate cyclase that mediates catabolite repression (Saier, 1989, 1993). In low-GC Gram-positive bacteria, cytoplasmic phosphorylated sugar metabolites are sensed by the HPr kinase which is allostericlaly activated. HPr becomes phosphorylated on Ser-46, and this phosphorylated derivative regulates the activities of its target proteins. These targets include (1) the PTS, (2) non-PTS permeases (both of which are inhibited) and (3) a cytoplasmic sugar-P phosphatase which is activated to reduce cytoplasmic inducer levels. Other important targets of HPr(ser-P) action are (4) the CcpA protein and probably (5) the CepB transcription factor. These two proteins together are believed to determine the intensity of catabolite repression. Their relative importance depends on physiological conditions. Both proteins may respond to the cytoplasmic concentration of HPr(ser-P) and appropriate metabolites. CepA possibly binds sugar metabolites such as FBP as well as HPr(ser-P). Because HPr(his-P, ser-P) does not bind to CepA, the regulatory cascade is also sensitive to the external PTS sugar concentration. Mutational analyses (unpublished results) suggest that CepA may bind to a site that includes His-15. Interestingly, both the CepA protein in the Gram-positive bacterium, B. subtilis, and glycerol kinase in the Gram-negative bacterium, E. coli, sense both a PTS protein and a cytoplasmic metabolic intermediate. The same may be true of target permeases and enzymes in both types of organisms, but this possibility has not yet been tested. The parallels between the Gram-negative and Gram-positive bacterial regulatory systems are superficial at the mechanistic level but fundamental at the functional level. Thus, the PTS participates in regulation in both cases, and phosphorylation of its protein constituents plays key roles. However, the stimuli sensed, the transmission mechanisms, the central PTS regulatory proteins that effect allosteric regulation, and some of the target proteins are completely different. It seems clear that these two transmission mechanisms evolved independently. They provide a prime example of functional convergence.

Two rpoN-linked ΔTn10-kan insertions suppress the conditionally lethal erats allele. One truncates rpoN while the second disrupts another gene (ptsN) in the rpoN operon and does not affect classical nitrogen regulation. Neither alter expression of era indicating that suppression is post-translational. Plasmid clones of ptsN prevent suppression by either disruption mutation indicating that this gene is important for lethality caused by erats. rpoN and six neighboring genes were sequenced and compared with sequences in the database. Two of these genes encode proteins homologous to Enzyme IIAFru and HPr of the phosphoenolpyruvate:sugar phosphotransferase system. We designate these proteins IIANtr (ptsN) and NPr (npr). Purified IIANtr and NPr exchange phosphate appropriately with Enzyme I, HPr, and Enzyme IIA proteins of the phosphoenolpyruvate:sugar phosphotransferase system. Several sugars and tricarboxylic acid cycle intermediates inhibited growth of the ptsN disruption mutant on medium containing an amino acid or nucleoside base as a combined source of nitrogen, carbon, and energy. This growth inhibition was relieved by supplying the ptsN gene or ammonium salts but was not aleviated by altering levels of exogenously supplied cAMP. These results support our previous proposal of a novel mechanism linking carbon and nitrogen assimilation and relates IIANtr to the unknown process regulated by the essential GTPase Era. Two rpoN-linked ΔTn10-kan insertions suppress the conditionally lethal erats allele. One truncates rpoN while the second disrupts another gene (ptsN) in the rpoN operon and does not affect classical nitrogen regulation. Neither alter expression of era indicating that suppression is post-translational. Plasmid clones of ptsN prevent suppression by either disruption mutation indicating that this gene is important for lethality caused by erats. rpoN and six neighboring genes were sequenced and compared with sequences in the database. Two of these genes encode proteins homologous to Enzyme IIAFru and HPr of the phosphoenolpyruvate:sugar phosphotransferase system. We designate these proteins IIANtr (ptsN) and NPr (npr). Purified IIANtr and NPr exchange phosphate appropriately with Enzyme I, HPr, and Enzyme IIA proteins of the phosphoenolpyruvate:sugar phosphotransferase system. Several sugars and tricarboxylic acid cycle intermediates inhibited growth of the ptsN disruption mutant on medium containing an amino acid or nucleoside base as a combined source of nitrogen, carbon, and energy. This growth inhibition was relieved by supplying the ptsN gene or ammonium salts but was not aleviated by altering levels of exogenously supplied cAMP. These results support our previous proposal of a novel mechanism linking carbon and nitrogen assimilation and relates IIANtr to the unknown process regulated by the essential GTPase Era.

This chapter discusses the utility of computer techniques, approached from the rationale of molecular biologists, for the correction of sequencing and assignment errors and for gleaning maximal information from DNA sequence data. Computer-aided approaches facilitate proper identification and characterization of operon, gene, and protein structures. The use of these approaches is exemplified in the chapter, drawing on a large body of representative published data concerned primarily with operons, including genes encoding proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) and other transport systems. The human element implies that genome sequences will be riddled with errors, errors that can be recognized when the appropriate tools are applied. The human brain lacks many of the capacities of a computer. Although the computer is an indispensible helpmate for searching and comparing sequences, biochemists and molecular biologists must guard against the blind acceptance of any sophisticated algorithmic output.

Bacteria impose regulatory mechanisms on metabolic processes to ensure that the needs of the cell are met but not exceeded. Here, we discuss the basic features of a mechanism by which carbohydrate catabolism in Gram-positive bacteria is regulated. Although the physiological consequences of this regulation are the same as in Gram-negative bacteria, the mechanism is entirely different. These regulatory processes evidently evolved late, after the divergence of Gram-positive from Gramnegative bacteria, even though the targets of regulation are universal.

Enzyme IIA(glc) and HPr are central regulatory and phosphocarrier proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) of bacteria. During phosphoryl transfer from phosphoenolpyruvate to glucose, phosphate is transferred from HPr to enzyme IIA(glc). In order to characterize the binding interfaces of the two proteins during phosphate transfer, N-15-edited and N-15-filtered NMR experiments have been recorded for the complex of enzyme IIA(glc) and HPr from Bacillus subtilis. Uniformly N-15-labeled enzyme IIA(glc) and nonlabeled HPr were used in these studies. Residues which undergo significant chemical shift changes upon complex formation have been identified for both proteins. The binding interfaces of the two proteins, suggested by the observed chemical shift changes, involve predominantly hydrophobic surfaces near the active site His-15 of HPr and the phosphoryl acceptor His-83 of IIA(glc).

The present work evaluated polyphosphate (poly P) metabolism in nuclear and mitochondrial fractions during Rhipicephalus microplus embryogenesis. Nuclear poly P decreased and activity of exopolyphosphatase (PPX — polyphosphate–phosphohydrolases; EC 3.6.1.11) increased after embryo cellularization until the end of embryogenesis. The utilization of mitochondrial poly P content occurred between embryo cellularization and segmentation stages. Increasing amounts of total RNA extracted from eggs progressively enhanced nuclear PPX activity, whereas it exerted no effect on mitochondrial PPX activity. The decline in total poly P content after the 7th day of embryogenesis does not reflect the free Pi increase and the total poly P chain length decrease after embryo cellularization. The Kmapp utilizing poly P3, poly P15 and poly P65 as substrate was almost the same for the nuclear fraction (around 1μM), while the affinity for substrate in mitochondrial fraction was around 10 times higher for poly P3 (Kmapp = 0.2μM) than for poly P15 (Kmapp = 2.8μM) and poly P65 (Kmapp = 3.6μM). PPX activity was stimulated by a factor of two by Mg2+ and Co2+ in the nuclear fraction and only by Mg2+ in the mitochondrial fraction. Heparin (20µg/mL) inhibited nuclear and mitochondrial PPX activity in about 90 and 95% respectively. Together, these data are consistent with the existence of two different PPX isoforms operating in the nuclei and mitochondria of the hard tick R. microplus with distinct metal dependence, inhibitor and activator sensitivities. The data also shed new light on poly P biochemistry during arthropod embryogenesis, opening new routes for future comparative studies on the physiological roles of different poly P pools distributed over cell compartments.

Sugar uptake and cytoplasmic inducer generation appear to be regulated by the phosphenolpyruvate: sugar phosphotransferase system (PTS) by distinct mechanisms in Gram-negative versus Grampositive bacteria. In Gram-negative bacteria, the free form of the glucose-specific IIA protein of the PTS, which can be phosphorylated on a histidyl residue by PEP and the PTS energy coupling proteins, inhibits non-PTS permease activities. In Gram-positive bacteria, the phosphorylated form of the energy coupling HPr protein of the PTS, which can, be phosphorylated on a seryl residue by ATP and a protein kinase, appears to inhibit non-PTS permease activities. In this summary article, the current status of these two PTS-mediated regulatory mechanisms will be evaluated.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTAssignment of the aliphatic proton and carbon-13 resonances of the Bacillus subtilis glucose permease IIA domain using double- and triple-resonance heteronuclear three-dimensional NMR spectroscopyWayne J. Fairbrother, Arthur G. Palmer, III, Mark Rance, Jonathan Reizer, Milton H. Saier, Jr., and Peter E. WrightCite this: Biochemistry 1992, 31, 18, 4413–4425Publication Date (Print):May 12, 1992Publication History Published online1 May 2002Published inissue 12 May 1992https://pubs.acs.org/doi/10.1021/bi00133a005https://doi.org/10.1021/bi00133a005research-articleACS PublicationsRequest reuse permissionsArticle Views74Altmetric-Citations39LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts

Nearly complete assignment of the aliphatic H-1 and C-13 resonances of the IIA(glc) domain of Bacillus subtilis has been achieved using a combination of double- and triple-resonance three-dimensional (3D) NMR experiments. A constant-time 3D triple-resonance HCA(CO)N experiment, which correlates the H-1(alpha) and C-13(alpha) chemical shifts of one residue with the amide N-15 chemical shift of the following residue, was used to obtain sequence-specific assignments of the C-13(alpha) resonances. The H-1(alpha) and amide N-15 chemical shifts had been sequentially assigned previously using principally 3D H-1-N-15 NOESY-HMQC and TOCSY-HMQC experiments [Fairbrother, W. J., Cavanagh, J., Dyson, H. J., Palmer, A. G., III, Sutrina, S. L., Reizer, J., Saier, M. H., Jr., & Wright, P. E. (1991) Biochemistry 30, 6896-69071. The side-chain spin systems were identified using 3D HCCH-COSY and HCCH-TOCSY spectra and were assigned sequentially on the basis of their H-1(alpha) and C-13(alpha) chemical shifts. The 3D HCCH and HCA(CO)N experiments rely on large heteronuclear one-bond J couplings for coherence transfers and therefore offer a considerable advantage over conventional H-1-H-1 correlation experiments that rely on H-1-H-1 3J couplings, which, for proteins the size of IIA(glc) (17.4 kDa), may be significantly smaller than the H-1 line widths. The assignments reported herein are essential for the determination of the high-resolution solution structure of the IIA(glc) domain of B. subtilis using 3D and 4D heteronuclear edited NOESY experiments; these assignments have been used to analyze 3D H-1-N-15 NOESY-HMQC and H-1-C-13 NOESY-HSQC spectra and calculate a low-resolution structure [Fairbrother, W. J., Gippert, G. P., Reizer, J., Saier, M. H., Jr., & Wright, P. E. (1992) FEBS Lett. 296, 148-152].

The phosphohydrolysis properties of the following phosphoprotein intermediates of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) were investigated: enzyme I, HPr, and the IIAGlc domain of the glucose enzyme II of Bacillus subtilis; and IIAGlc (fast and slow forms) of Escherichia coli. The phosphohydrolysis properties were also studied for the site-directed mutant H68A of B. subtilis IIA Glc. Several conclusions were reached. (i) The phosphohydrolysis properties of the homologous phosphoprotein intermediates of B. subtilis and E. coli are similar. (ii) These properties deviate from those of isolated N delta 1- and N epsilon 2-phosphohistidine indicating the participation of neighbouring residues at the active sites of these proteins. (iii) The rates of phosphohydrolysis of the H68A mutant of B. subtilis IIAGlc were reduced compared with the wild-type protein, suggesting that both His-83 and His-68 are present at the active site of wild-type IIAGlc. (iv) The removal of seven N-terminal residues of E. coli IIAGlc reduced the rates of phosphohydrolysis between pH 5 and 8.

Two monovalent ion porters, the putative Na+/H+ antiporter (NapA) of Enterococcus hirae and the putative K+/H+ antiporter (KefC) of Escherichia coli, are similar in sequence throughout their hydrophobic domains. These two proteins, which comprise a novel family of transporters unrelated to the previously characterized Na+/H+ exchangers of E. coli (NhaA and NhaB) are proposed to function by essentially the same mechanism.

Several classes of transport systems function by different mechanisms, couple different forms of energy to transmembrane solute translocation and comprise distinct families of homologous proteins. Some of these permease classes, most of which extend across the prokaryotic-eukaryotic boundary, share structural and functional characteristics which suggest that they form a superfamily, i.e. share a common evolutionary origin.

Phosphorylation of a major 57-kilodalton protein substrate was observed in cell lysates of Spiroplasma melliferum BC3 incubated with [gamma-32P]ATP. Only serine phosphates have been isolated from the acid hydrolysate of the phosphorylated protein. The 57-kilodalton protein substrate was found, to a large extent, in the cytosolic fraction and, to a lesser extent, associated with cell membranes and was detected in the Triton X-100-insoluble fraction that contained fibrils.

This review consists of three major sections. The first and largest section reviews the protein constituents and known properties of the phosphotransferase systems present in well-studied Gram-positive bacteria. These bacteria include species of the following genera: (1) Staphylococcus, (2) Streptococcus, (3) Bacillus, (4) Lactobacillus, (5) Clostridium, (6) Arthrobacter, and (7) Brochothrix. The properties of the different systems are compared. The second major section deals with the regulation of carbohydrate uptake. There are four parts: (1) inhibition by intracellular sugar phosphates in Staphylococcus aureus, (2) PTS-mediated regulation of glycerol uptake in Bacillus subtilis, (3) competition for phospho-HPr in Streptococcus mutans, and (4) the possible involvement of protein kinases in the regulation of sugar uptake via the phosphotransferase system. The third section deals with the phenomenon of inducer expulsion. The first part is concerned with the physiological characterization of the phenomenon; then the consequences of unregulated uptake and expulsion, a futile cycle of energy expenditure, are considered. Finally, the biochemistry of the protein kinase and the protein phosphate phosphatase system, which appears to regulate sugar transport via the phosphotransferase system, is defined. The review, therefore, concentrates on the phosphotransferase system, its functions in carbohydrate transport and phosphorylation, the mechanisms of its regulation, and the mechanism by which it participates in the regulation of other physiological processes in the bacterial cell.