Jörg Stülke

Carbon catabolite repression in bacteria: many ways to make the most out of nutrients.

Most bacteria can selectively use substrates from a mixture of different carbon sources. The presence of preferred carbon sources prevents the expression, and often also the activity, of catabolic systems that enable the use of secondary substrates. This regulation, called carbon catabolite repression (CCR), can be achieved by different regulatory mechanisms, including transcription activation and repression and control of translation by an RNA-binding protein, in different bacteria. Moreover, CCR regulates the expression of virulence factors in many pathogenic bacteria. In this Review, we discuss the most recent findings on the different mechanisms that have evolved to allow bacteria to use carbon sources in a hierarchical manner.

Carbon catabolite repression in bacteria.

Carbon catabolite repression (CCR) is a regulatory mechanism by which the expression of genes required for the utilization of secondary sources of carbon is prevented by the presence of a preferred substrate. This enables bacteria to increase their fitness by optimizing growth rates in natural environments providing complex mixtures of nutrients. In most bacteria, the enzymes involved in sugar transport and phosphorylation play an essential role in signal generation leading through different transduction mechanisms to catabolite repression. The actual mechanisms of regulation are substantially different in various bacteria. The mechanism of lactose-glucose diauxie in Escherichia coli has been reinvestigated and was found to be caused mainly by inducer exclusion. In addition, the gene encoding HPr kinase, a key component of CCR in many bacteria, was discovered recently.

PRD--a protein domain involved in PTS-dependent induction and carbon catabolite repression of catabolic operons in bacteria.

Several operon‐specific transcriptional regulators, including antiterminators and activators, contain a duplicated conserved domain, the PTS regulation domain (PRD). These duplicated domains modify the activity of the transcriptional regulators both positively and negatively. PRD‐containing regulators are very common in Gram‐positive bacteria. In contrast, antiterminators controlling β‐glucoside utilization are the only functionally characterized members of this family from Gram‐negative bacteria. PRD‐containing regulators are controlled by PTS‐dependent phosphorylation with different consequences: (i) In the absence of inducer, the phosphorylated EIIB component of the sugar permease donates its phosphate to a PRD, thereby inactivating the regulator. In the presence of the substrate, the regulator is dephosphorylated, and the phosphate is transferred to the sugar, resulting in induction of the operon. (ii) In Gram‐positive bacteria, a novel mechanism of carbon catabolite repression mediated by PRD‐containing regulators has been demonstrated. In the absence of PTS substrates, the HPr protein is phosphorylated by enzyme I at His‐15. This form of HPr can, in turn, phosphorylate PRD‐containing regulators and stimulate their activity. In the presence of rapidly metabolizable carbon sources, ATP‐dependent phosphorylation of HPr at Ser‐46 by HPr kinase inhibits phosphorylation by enzyme I, and PRD‐containing regulators cannot, therefore, be stimulated and are inactive. All regulators of this family contain two copies of PRD, which are functionally specialized in either induction or catabolite repression.

Temporal activation of β-glucanase synthesis in Bacillus subtilis is mediated by the GTP pool

beta-Glucanase synthesis was temporally activated in Bacillus subtilis at the onset of stationary phase. This regulation was dependent upon a drop in the GTP concentration in response to nutrient limitation. The Spo0A and AbrB proteins were involved in the GTP-dependent temporal activation.

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.

Induction of the Bacillus subtilis ptsGHI operon by glucose is controlled by a novel antiterminator, GlcT.

Glucose is the preferred carbon and energy source of Bacillus subtilis. It is transported into the cell by the glucose‐specific phosphoenolpyruvatesugar phosphotransferase system (PTS) encoded by the ptsGHI locus. We show here that these three genes (ptsG, ptsH, and ptsI ) form an operon, the expression of which is inducible by glucose. In addition, ptsH and ptsI form a constitutive ptsHI operon. The promoter of the ptsGHI operon was mapped and expression from this promoter was found to be constitutive. Deletion mapping of the promoter region revealed the presence of a transcriptional terminator as a regulatory element between the promoter and coding region of the ptsG gene. Mutations within the ptsG gene were characterized and their consequences on the expression of ptsG studied. The results suggest that expression of the ptsGHI operon is subject to negative autoregulation by the glucose permease, which is the ptsG gene product. A regulatory gene located upstream of the ptsGHI operon, termed glcT, was also identified. The GlcT protein is a novel member of the BglG family of transcriptional antiterminators and is essential for the expression of the ptsGHI operon. A deletion of the terminator alleviates the need for GlcT. The activity of GlcT is negatively regulated by the glucose permease.

Cross-talk between phosphorylation and lysine acetylation in a genome-reduced bacterium

Protein post‐translational modifications (PTMs) represent important regulatory states that when combined have been hypothesized to act as molecular codes and to generate a functional diversity beyond genome and transcriptome. We systematically investigate the interplay of protein phosphorylation with other post‐transcriptional regulatory mechanisms in the genome‐reduced bacterium Mycoplasma pneumoniae. Systematic perturbations by deletion of its only two protein kinases and its unique protein phosphatase identified not only the protein‐specific effect on the phosphorylation network, but also a modulation of proteome abundance and lysine acetylation patterns, mostly in the absence of transcriptional changes. Reciprocally, deletion of the two putative N‐acetyltransferases affects protein phosphorylation, confirming cross‐talk between the two PTMs. The measured M. pneumoniae phosphoproteome and lysine acetylome revealed that both PTMs are very common, that (as in Eukaryotes) they often co‐occur within the same protein and that they are frequently observed at interaction interfaces and in multifunctional proteins. The results imply previously unreported hidden layers of post‐transcriptional regulation intertwining phosphorylation with lysine acetylation and other mechanisms that define the functional state of a cell.

Novel phosphotransferase system genes revealed by genome analysis - the complete complement of PTS proteins encoded within the genome of Bacillus subtilis

Bacillus subtilis can utilize several sugars as single sources of carbon and energy. Many of these sugars are transported and concomitantly phosphorylated by the phosphoenolpyruvate:sugar phosphotransferase system (PTS). In addition to its role in sugar uptake, the PTS is one of the major signal transduction systems in B. subtilis. In this study, an analysis of the complete set of PTS proteins encoded within the B. subtilis genome is presented. Fifteen sugar-specific PTS permeases were found to be present and the functions of novel PTS permeases were studied based on homology to previously characterized permeases, analysis of the structure of the gene clusters in which the permease encoding genes are located and biochemical analysis of relevant mutants. Members of the glucose, sucrose, lactose, mannose and fructose/mannitol families of PTS permeases were identified. Interestingly, nine pairs of IIB and IIC domains belonging to the glucose and sucrose permease families are present in B. subtilis; by contrast only five Enzyme IIA(Glc)-like proteins or domains are encoded within the B. subtilis genome. Consequently, some of the EIIA(Glc)-like proteins must function in phosphoryl transfer to more than one IIB domain of the glucose and sucrose families. In addition, 13 PTS-associated proteins are encoded within the B. subtilis genome. These proteins include metabolic enzymes, a bifunctional protein kinase/phosphatase, a transcriptional cofactor and transcriptional regulators that are involved in PTS-dependent signal transduction. The PTS proteins and the auxiliary PTS proteins represent a highly integrated network that catalyses and simultaneously modulates carbohydrate utilization in this bacterium.

Trigger enzymes: bifunctional proteins active in metabolism and in controlling gene expression

All regulatory processes require components that sense the environmental or metabolic conditions of the cell, and sophisticated sensory proteins have been studied in great detail. During the last few years, it turned out that enzymes can control gene expression in response to the availability of their substrates. Here, we review four different mechanisms by which these enzymes interfere with regulation in bacteria. First, some enzymes have acquired a DNA‐binding domain and act as direct transcription repressors by binding DNA in the absence of their substrates. A second class is represented by aconitase, which can bind iron responsive elements in the absence of iron to control the expression of genes involved in iron homoeostasis. The third class of these enzymes is sugar permeases of the phosphotransferase system that control the activity of transcription regulators by phosphorylating them in the absence of the specific substrate. Finally, a fourth class of regulatory enzymes controls the activity of transcription factors by inhibitory protein–protein interactions. We suggest that the enzymes that are active in the control of gene expression should be designated as trigger enzymes. An analysis of the occurrence of trigger enzymes suggests that the duplication and subsequent functional specialization is a major pattern in their evolution.

The Phosphoproteome of the Minimal Bacterium Mycoplasma pneumoniae ANALYSIS OF THE COMPLETE KNOWN SER/THR KINOME SUGGESTS THE EXISTENCE OF NOVEL KINASES

Mycoplasma pneumoniae belongs to the Mollicutes, the group of organisms with the smallest genomes that are capable of host-independent life. These bacteria show little regulation in gene expression, suggesting an important role for the control of protein activities. We have studied protein phosphorylation in M. pneumoniae to identify phosphorylated proteins. Two-dimensional gel electrophoresis and mass spectrometry allowed the detection of 63 phosphorylated proteins, many of them enzymes of central carbon metabolism and proteins related to host cell adhesion. We identified 16 phosphorylation sites, among them 8 serine and 8 threonine residues, respectively. A phosphoproteome analysis with mutants affected in the two annotated protein kinase genes or in the single known protein phosphatase gene suggested that only one protein (HPr) is phosphorylated by the HPr kinase, HPrK, whereas four adhesion-related or surface proteins were targets of the protein kinase C, PrkC. A comparison with the phosphoproteomes of other bacteria revealed that protein phosphorylation is evolutionarily only poorly conserved. Only one single protein with an identified phosphorylation site, a phosphosugar mutase (ManB in M. pneumoniae), is phosphorylated on a conserved serine residue in all studied organisms from archaea and bacteria to man. We demonstrate that this protein undergoes autophosphorylation. This explains the strong conservation of this phosphorylation event. For most other proteins, even if they are phosphorylated in different species, the actual phosphorylation sites are different. This suggests that protein phosphorylation is a form of adaptation of the bacteria to the specific needs of their particular ecological niche.