In Vivo Activity of Enzymatic and Regulatory Components of the Phosphoenolpyruvate:Sugar Phosphotransferase System in Mycoplasma pneumoniae

Abstract

Mycoplasma pneumoniae is a pathogenic bacterium that is highly adapted to life on mucosal surfaces. This adaptation is reflected by the very compact genome and the small number of regulatory proteins. However, M. pneumoniae possesses the HPr kinase/phosphorylase (HPrK/P), the key regulator of carbon metabolism in the Firmicutes. In contrast to the enzymes of other bacteria, the HPrK/P of M. pneumoniae is already active at very low ATP concentrations, suggesting a different mode of regulation. In this work, we studied the ability of M. pneumoniae to utilize different carbohydrates and their effects on the activity of the different phosphotransferase system (PTS) components. Glucose served as the best carbon source, with a generation time of about 30 h. Fructose and glycerol were also used but at lower rates and with lower yields. In contrast, M. pneumoniae is unable to use mannitol even though the bacterium is apparently equipped with all the genes required for mannitol catabolism. This observation is probably a reflection of the continuing and ongoing reduction of the M. pneumoniae genome. The general enzymatic and regulatory components of the PTS, i.e., enzyme I, HPr, and HPrK/P, were present under all growth conditions tested in this study. However, HPrK/P activity is strongly increased if the medium contains glycerol. Thus, the control of HPrK/P in vivo differs strongly between M. pneumoniae and the other Firmicutes. This difference may relate to the specific conditions on lipid-rich cell surfaces.Mycoplasma pneumoniae is a pathogen that lives on mucosal surfaces and causes diseases such as mild pneumonia and tracheobronchitis and also causes complications affecting the central nervous system, the skin, and mucosal surfaces (19,24). The parasitic lifestyle of this bacterium is reflected by its small and highly compacted genome, its slow growth, and its reduced metabolic abilities. With only nine regulatory proteins, M. pneumoniae belongs to the organisms with the lowest number of regulators studied so far, suggesting a good adaptation to constant environments (5,15,37). In addition to regulatory proteins that are thought to act at the DNA level, we identified the key regulatory protein of carbon metabolism in grampositive bacteria, HPr kinase/phosphorylase (HPrK/P), in M. pneumoniae (33,42). Moreover, HPrK/P activity was detected in other Mollicutes such as Mycoplasma capricolum, Mycoplasma genitalium, and Acholeplasma laidlawii (17, 53).HPrK/P controls the activity of the HPr protein of the bacterial phosphoenolpyruvate:sugar phosphotransferase system by phosphorylation at a regulatory site, Ser-46. In the grampositive model organism Bacillus subtilis, this phosphorylation interferes with the phosphoenolpyruvate (PEP)-and enzyme I-dependent phosphorylation on His-15 of HPr, which is important for the phosphorylation of transported sugars (12,38). In addition to its role in sugar transport, HPr is the major signal transducer in carbon metabolism in low-GC gram-positive bacteria (now referred to as Firmicutes ...

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

“…M. pneumoniae HprK/P is unusual, as it can phosphorylate HPr at relatively low ATP concentrations (831). In addition, in vivo P-Ser-HPr formation is stimulated by the presence of glycerol in the growth medium (307). Although the M. pneumoniae enzyme was reported to dephosphorylate P-Ser-HPr in vitro in a P idependent manner, similar to HprK/Ps from other organisms (831), the P-Ser-HPr dephosphorylation activity exhibited by PrpC seems to be of physiological relevance.…”

“…Because in many hprK-containing gram-negative bacteria, yhbJ and other genes of the E. coli rpoN region are organized in an operon with hprK, it is tempting to assume that Crh might somehow play a role in the regulation of SigL ( 54 ) (308). The role of P-Ser-HPr formation in these organisms therefore remains unknown (307).…”

Comparative Genomic Analyses of the Bacterial Phosphotransferase System

“…The regulation of HprK in the Mollicutes may be different from that in other Firmicutes (26). The distribution and numbers of PTS permeases in the Mollicutes also differ from those in other Firmicutes.…”

The acidic, glutamine-rich Mpn474 protein of Mycoplasma pneumoniae is surface exposed and covers the complete cell

“…For Western blot analysis, M. pneumoniae crude cell extracts (Halbedel et al, 2004) were separated on 10 % SDS-polyacrylamide gels. After electrophoresis, the proteins were transferred to a PVDF membrane (Bio-Rad) by electroblotting.…”