Jonathan Reizer

Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen.

Pseudomonas aeruginosa is a ubiquitous environmental bacterium that is one of the top three causes of opportunistic human infections. A major factor in its prominence as a pathogen is its intrinsic resistance to antibiotics and disinfectants. Here we report the complete sequence of P. aeruginosa strain PAO1. At 6.3 million base pairs, this is the largest bacterial genome sequenced, and the sequence provides insights into the basis of the versatility and intrinsic drug resistance of P. aeruginosa. Consistent with its larger genome size and environmental adaptability, P. aeruginosa contains the highest proportion of regulatory genes observed for a bacterial genome and a large number of genes involved in the catabolism, transport and efflux of organic compounds as well as four potential chemotaxis systems. We propose that the size and complexity of the P. aeruginosa genome reflect an evolutionary adaptation permitting it to thrive in diverse environments and resist the effects of a variety of antimicrobial substances.

The vacuolar membrane protein gamma-TIP creates water specific channels in Xenopus oocytes.

The vacuolar membrane (tonoplast) of higher plant cells contains an abundant 27 kDa protein called TIP (tonoplast intrinsic protein) that occurs in different isoforms and belongs to a large family of homologous channel‐like proteins found in bacteria, plants and animals. In the present study, we identified and characterized the function of gamma‐TIP from Arabidopsis thaliana by expression of the protein in Xenopus oocytes. gamma‐TIP increased the osmotic water permeability of oocytes 6‐ to 8‐fold, to values in the range 1–1.5 × 10(−2) cm/s. Similar results were obtained with the homologous human erythrocyte protein CHIP28, recently identified as the erythrocyte water channel. The bacterial homolog GlpF did not affect the osmotic water permeability of oocytes, but facilitated glycerol uptake, in accordance with its known function. By contrast, gamma‐TIP did not promote glycerol permeability. Voltage clamp experiments provided evidence showing that gamma‐TIP induced no electrogenic ion transport in oocytes, especially during osmotic challenge that resulted in massive transport of water. These results allow us to conclude that the various protein members of the MIP family have unique and specific transport functions and that the plant protein gamma‐TIP likely functions as a water specific channel in the vacuolar membrane.

Two novel families of bacterial membrane proteins concerned with nodulation, cell division and transport.

Homology has been established for members of two families of functionally related bacterial membrane proteins. The first family (the resistance/nodulation/cell division (RND) family) Includes (i) two metal‐resistance efflux pumps in Alcaligenes eutrophus (CzcA and CnrA), (ii) three proteins which function together in nodulation of alfalfa roots by Rhizobium meliloti (NoIGHI), and (iii) a cell division protein in Escherichia coli (EnvD). The second family (the putative membrane fusion protein (MFP) family) includes a nodulation protein (NoIF), a cell division protein (EnvC), and a multidrug resistance transport protein (EmrA). We propose that an MFP functions co‐operatively with an RND protein to transport large or hydrophobic molecules across the two membranes of the Gram‐negative bacterial cell envelope.

The MIP Family of Integral Membrane Channel Proteins: Sequence Comparisons, Evolutionary Relationships, Reconstructed Pathway of Evolution, and Proposed Functional Differentiation of the Two Repeated Halves of the Proteins

AbstractThe major intrinsic protein (MIP) of the bovine lens fiber cell membrane was the first member of the MIP family of proteins to be sequenced and characterized. It is probably a homotetramer with transmembrane channel activity that plays a role in lens biogenesis or maintenance. The polypeptide chain of each subunit may span the membrane six times, and both the N- and C-termini face the cell cytoplasm. Eighteen sequenced or partially sequenced proteins from bacteria, yeast, plants, and animals have now been shown to be members of the MIP family. These proteins appear to function in (1) metazoan development and neurogenesis (MIP and BIB), (2) water transport across the human erythrocyte membrane (ChIP), (3) communication between host plant cells and symbiotic nitrogen-fixing bacteria (NOD), (4) transport across the tonoplast membrane during plant seed development (α-TIP), (5) water stress-induced resistance to desiccation in plants (Wsi-TIP), (6) suppression of a genetic growth defect on fermentable ...

Catabolite repression and inducer control in Gram-positive bacteria

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.

A novel protein kinase that controls carbon catabolite repression in bacteria

HPr(Ser) kinase is the sensor in a multicomponent phosphorelay system that controls catabolite repression, sugar transport and carbon metabolism in Gram‐positive bacteria. Unlike most other protein kinases, it recognizes the tertiary structure in its target protein, HPr, a phosphocarrier protein of the bacterial phosphotransferase system and a transcriptional cofactor controlling the phenomenon of catabolite repression. We have identified the gene (ptsK) encoding this serine/threonine protein kinase and characterized the purified protein product. Orthologues of PtsK have been identified only in bacteria. These proteins constitute a novel family unrelated to other previously characterized protein phosphorylating enzymes. The Bacillus subtilis kinase is shown to be allosterically activated by metabolites such as fructose 1,6‐bisphosphate and inhibited by inorganic phosphate. In contrast to wild‐type B. subtilis, the ptsK mutant is insensitive to transcriptional regulation by catabolite repression. The reported results advance our understanding of phosphorylation‐dependent carbon control mechanisms in Gram‐positive bacteria.

The bacterial phosphotransferase system: new frontiers 30 years later

In 1964, Kundig, Ghosh and Roseman reported the discovery of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Thirty years later, we find that the PTS functions not only as a sugar‐phosphorylating system, but also as a complex protein kinase system that regulates a wide variety of metabolic processes and controls the expression of numerous genes. As a result of recent operon‐ and genome‐sequencing projects, novel PTS protein‐encoding genes have been discovered, most of which have yet to be functionally defined. Some of them appear to be involved in cellular processes distinct from those recognized previously. Fundamental aspects of past and current PTS research are briefly reviewed, and recent advances are integrated into conceptual pictures that provide guides for future research.

A functional superfamily of sodium/solute symporters

Eleven families of sodium/solute symporters are defined based on their degrees of sequence similarities, and the protein members of these families are characterized in terms of their solute and cation specificities, their sizes, their topological features, their evolutionary relationships, and their relative degrees and regions of sequence conservation. In some cases, particularly where site-specific mutagenesis analyses have provided functional information about specific proteins, multiple alignments of members of the relevant families are presented, and the degrees of conservation of the mutated residues are evaluated. Signature sequences for several of the eleven families are presented to facilitate identification of new members of these families as they become sequenced. Phylogenetic tree construction reveals the evolutionary relationships between members of each family. One of these families is shown to belong to the previously defined major facilitator superfamily. The other ten families do not show sufficient sequence similarity with each other or with other identified transport protein families to establish homology between them. This study serves to clarify structural, functional and evolutionary relationships among eleven distinct families of functionally related transport proteins.

Evolutionary relationships between sugar kinases and transcriptional repressors in bacteria

We have characterized a new family of proteins (the ROK family) which includes six transcriptional repressors for sugar catabolic operons, three sugar kinases, and three unidentified open reading frames. Analysis of the aligned sequences and phylogenetic tree construction allow predictions regarding the functional nature of conserved domains and residues within these proteins as well as the pathway of evolutionary divergence that gave rise to the family.

The Phosphoenolpyruvate:Sugar Phosphotransferase System in Gram-Positive Bacteria: Properties, Mechanism, and Regulation

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.