Searching the genome sequence of Streptococcus pneumoniae revealed the presence of a single Ser/Thr protein kinase gene stkP linked to protein phosphatase phpP. Biochemical studies performed with recombinant StkP suggest that this protein is a functional eukaryotic‐type Ser/Thr protein kinase. In vitro kinase assays and Western blots of S. pneumoniae subcellular fractions revealed that StkP is a membrane protein. PhpP is a soluble protein with manganese‐dependent phosphatase activity in vitro against a synthetic substrate RRA(pT)VA. Mutations in the invariant aspartate residues implicated in the metal binding completely abolished PhpP activity. Autophosphorylated form of StkP was shown to be a substrate for PhpP. These results suggest that StkP and PhpP could operate as a functional pair in vivo. Analysis of phosphoproteome maps of both wild‐type and stkP null mutant strains labeled in vivo and subsequent phosphoprotein identification by peptide mass fingerprinting revealed two possible substrates for StkP. The evidence is presented that StkP can phosphorylate in vitro phosphoglucosamine mutase GlmM which catalyzes the first step in the biosynthetic pathway leading to the formation of UDP‐N‐acetylglucosamine, an essential common precursor to cell envelope components.

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SummaryBacteria typically undergo intermittent periods of starvation and adaptation, emulated as diauxic growth in the laboratory. In association with growth arrest elicited by metabolic stress, the differentiating eubacterium Streptomyces coelicolor not only adapts its primary metabolism, but can also activate developmental programmes leading to morphogenesis and antibiotic biosynthesis. Here, we report combined proteomic and metabolomic data of S. coelicolor used to analyse global changes in gene expression during diauxic growth in a defined liquid medium. Cultures initially grew on glutamate, providing the nitrogen source and feeding carbon (as 2-oxoglutarate) into the TCA cycle, followed by a diauxic delay allowing reorientation of metabolism and a second round of growth supported by NH 4 + + + + , formed during prediauxic phase, and maltose, a glycolytic substrate. Cultures finally entered stationary phase as a result of nitrogen starvation. These four physiological states had previously been defined statistically by their distinct patterns of protein synthesis and heat shock responses. Together, these data demonstrated that the rates of synthesis of heat shock proteins are determined not only by temperature increase but also by the patterns and rates of metabolic flux in certain pathways. Synthesis profiles for metabolic-and stress-induced proteins can now be interpreted by the identification of 204 spots (SWICZ database presented at http:// proteom.biomed.cas.cz). Cluster analysis showed that the activity of central metabolic enzymes involved in glycolysis, the TCA cycle, starvation or proteolysis each displayed identifiable patterns of synthesis that logically underlie the metabolic state of the culture. Diauxic lag was accompanied by a structured regulatory programme involving the sequential activation of heat-, salt-, cold-and bacteriostatic antibiotic (pristinamycin I, PI)-induced stimulons. Although stress stimulons presumably provide protection during environmental-or starvationinduced stress, their identities did not reveal any coherent adaptive or developmental functions. These studies revealed interactive regulation of metabolic and stress response systems including some proteins known to support developmental programmes in S. coelicolor .

Chronic hypoxia induces lung vascular remodeling, which results in pulmonary hypertension. We hypothesized that a previously found increase in collagenolytic activity of matrix metalloproteinases during hypoxia promotes pulmonary vascular remodeling and hypertension. To test this hypothesis, we exposed rats to hypoxia (fraction of inspired oxygen = 0.1, 3 wk) and treated them with a metalloproteinase inhibitor, Batimastat (30 mg/kg body wt, daily ip injection). Hypoxia-induced increases in concentration of collagen breakdown products and in collagenolytic activity in pulmonary vessels were inhibited by Batimastat, attesting to the effectiveness of Batimastat administration. Batimastat markedly reduced hypoxic pulmonary hypertension: pulmonary arterial blood pressure was 32 +/- 3 mmHg in hypoxic controls, 24 +/- 1 mmHg in Batimastat-treated hypoxic rats, and 16 +/- 1 mmHg in normoxic controls. Right ventricular hypertrophy and muscularization of peripheral lung vessels were also diminished. Batimastat had no influence on systemic arterial pressure or cardiac output and was without any effect in rats kept in normoxia. We conclude that stimulation of collagenolytic activity in chronic hypoxia is a substantial causative factor in the pathogenesis of pulmonary vascular remodeling and hypertension.

Hypoxic pulmonary vasoconstriction (HPV) is a beneficial mechanism that diverts blood from hypoxic alveoli to better ventilated areas of the lung, but breathing hypoxic air causes the pulmonary circulation to become hypertensive. Responses to airway hypoxia are associated with depolarization of smooth muscle cells in the pulmonary arteries and reduced activity of K+ channels. As Kv7 channels have been proposed to play a key role in regulating the smooth muscle membrane potential, we investigated their involvement in the development of HPV and hypoxia-induced pulmonary hypertension. Vascular effects of the selective Kv7 blocker, linopirdine, and Kv7 activator, flupirtine, were investigated in isolated, saline-perfused lungs from rats maintained for 3–5 days in an isobaric hypoxic chamber (FiO2 = 0.1) or room air. Linopirdine increased vascular resistance in lungs from normoxic, but not hypoxic rats. This effect was associated with reduced mRNA expression of the Kv7.4 channel α-subunit in hypoxic arteries, whereas Kv7.1 and Kv7.5 were unaffected. Flupirtine had no effect in normoxic lungs but reduced vascular resistance in hypoxic lungs. Moreover, oral dosing with flupirtine (30 mg/kg/day) prevented short-term in vivo hypoxia from increasing pulmonary vascular resistance and sensitizing the arteries to acute hypoxia. These findings suggest a protective role for Kv7.4 channels in the pulmonary circulation, limiting its reactivity to pressor agents and preventing hypoxia-induced pulmonary hypertension. They also provide further support for the therapeutic potential of Kv7 activators in pulmonary vascular disease.