The opg mutants exhibit a pleiotropic phenotype. Colonies of opg mutants are mucoid, indicating increased biosynthesis of EPS. This correlates with a threefold increase in the expression of the epsG::lacZ fusion in an opgG mutant background. This increase could be the result of a direct action of OPGs on eps gene transcription via a regulatory protein(s). Actually, in E. coli, the cps genes, involved in colanic acid capsular polysaccharide biosynthesis, are regulated by a two-component sensor-regulator system, RcsB/RcsC. In an mdoH mutant, cps gene transcription is increased but remains regulated in an RcsB/RcsC-dependent fashion. This suggests that RcsC, the sensor of the two-component system, could sense the level of OPG in the periplasm in a still unknown way (). In Erwinia amylovora and Erwinia stewartii, the RcsB and RcsC proteins are involved in the regulation of EPS biosynthesis and show strong similarities with the RcsB and RscC proteins of E. coli, indicating the existence of a family of related capsule activator proteins (). Thus, in E. chrysanthemi, OPGs could also participate in EPS biosynthesis regulation by acting via a similar two-component regulatory system. Alternatively, this increase could be one of the responses resulting from a disorganization of the cell envelope induced by the lack of OPGs. Disorganization of the cell envelope is suggested by the other phenotypes of the opg mutants, particularly bile salt hypersensivity and defective chemotaxis. Lack of OPGs could result in abnormal assembly of several envelope components due to an unknown structural function of the glucans, or OPG concentration could be detected and used to regulate the synthesis of several envelope components, especially those which are subjected to osmotic regulation, like the flagellum apparatus () and colanic acid synthesis () in E. coli.
Glycerol can be phosphorylated in a GlpK-catalyzed, ATP-dependent mechanism, yielding glycerol-3-phosphate (). Alternatively, GldA (glycerol dehydrogenase) can convert glycerol to glycerone in an NAD+-dependent reaction (). The decreased GldA and increased GlpK levels in the pigX mutant suggest that the level of glycerol-3-phosphate, which may be funneled into either glycolysis or phospholipid biosynthesis, will be elevated.
Nitrogen source: In most microorganisms, both inorganic and organic forms of nitrogen are metabolized to produce amino acids, nucleic acids, proteins, and cell wall components 69. The fibrinolytic protease production is dependent on the availability of both carbon and nitrogen sources in the medium 68. Although complex nitrogen sources are usually used for fibrinolytic protease production, the requirement for a specific nitrogen supplement differs from organism to organism. Low levels of fibrinolytic protease production were reported with the use of inorganic nitrogen sources in the production medium 43, 68, 70.
Previously, the prodigiosin master regulator, PigP, was shown to negatively affect the expression of the pigX gene. Hyperpigmented random transposon mutant strains (ROP4 and ROP4S) and a decreased pigment production mutant strain (HSPIG66) were identified in a screen for regulators of Pig biosynthesis and were mapped either to within pigX (ROP4 and ROP4S) or 5′ of pigX (HSPIG66) (). The sequence and genomic context of pigX were completed and are depicted schematically in Fig. . The 649-amino-acid (aa) predicted PigX protein is similar to YhdA from E. coli (64% similarity/54% identity) and is most closely related to the predicted product of ECA0266 from Erwinia carotovora subsp. atroseptica SCRI1043 (80% similarity/73% identity). Divergently transcribed from and 5′ of pigX is a 984-bp open reading frame, which is predicted to encode a homolog of YhdH from E. coli (76% similarity/69% identity), a putative quinone oxidoreductase. PigX is predicted to have two N-terminal transmembrane helices and a central GGDEF domain (aa 222 to 384), as well as a C-terminal EAL domain (aa 398 to 633) (Fig. ). Therefore, it was predicted that PigX might be involved in intracellular c-di-GMP metabolism.
Enzyme synthesis was found to be repressed by rapidly metabolizable nitrogen sources such as amino acids or ammonium ion concentrations in the medium 22, 24. However, one report indicated no repression in the fibrinolytic protease activity with the use of ammonium salts 43. An increase in fibrinolytic protease production by the addition of ammonium sulphate was also observed by Amrita Raja and Nancy Khess 43. Similarly, sodium nitrate was found to be stimulatory for fibrinolytic protease production 43. Substitution of silver nitrate in the basal medium with sodium nitrate increased enzyme production even more 71.
Characterization of FMP Iron Pool—The 59Fe-labeled doublet detected on native gel was further resolved to reveal its individual polypeptides by denaturing SDS-PAGE as described under “Experimental Procedures” and in . We identified several spots, ranging from 3 to 10, depending on the experiment considered. The corresponding polypeptides were analyzed by mass spectrometry. These polypeptides were related to basic biological processes, such as protein translation and folding, energy/carbon metabolism, amino acid biosynthesis, and potential metal ion binding (supplemental Table S3). FMP iron may thus play a role in protein translation. Given that magnesium ions contribute to maintenance of the 50 S ribosomal architecture and is necessary for EF-Tu protein chain elongation activity (, ), we checked that ferrous ions were not replacing magnesium ions. Increasing the magnesium concentration in the culture medium up to 20 m, i.e. 0.5 m in Tris medium, did not affect formation of this pool (). We analyzed the effect of antibiotics - fusidic acid, which blocks EF-G-mediated translocation of peptidyl-tRNA on the ribosome, and tetracycline, which disrupts codon/anticodon interactions - on this iron pool (, ) (). These drugs were added to cultures 5 min before adding 59Fe-chrysobactin. Formation of the pool of FMP iron was severely disrupted. The continuum of bands was also strongly reduced, whereas the pool of iron bound to bacterioferritin remained unchanged. Nalidixic acid, an inhibitor of DNA gyrase (), had no effect. Thus, de novo protein biosynthesis is required for detection of 59Fe signals corresponding to FMP and to the continuum of protein bands.
A proteomic strategy was devised to identify cellular changes in the absence of a functional PigX (Fig. and Table ). As mentioned above, the levels of proteins implicated in the biosynthesis of Pig were elevated in the hyperpigmented mutant strain. Furthermore, proteins involved in amino acid biosynthesis and uptake were altered in the pigX mutant strain. Increased levels of SerC, involved in serine biosynthesis; PutA, a proline utilization membrane protein; and LivK, a periplasmic component of an ABC-type branched-chain amino acid transporter, were detected in the pigX mutant. In addition, the pigX mutant displayed decreased levels of AspA, an enzyme required for -aspartate metabolism (-aspartate is linked to serine and glycine biosynthesis) (). Serine and proline are precursors in the biosynthetic pathway of prodigiosin (). It is possible that the elevated level of SerC, which catalyzes the second step of serine biosynthesis (), provides increased serine for the overproduction of pigment in the pigX background. The multifunctional PutA flavoenzyme converts proline into glutamate in a two-step reaction and is also a DNA binding protein that represses its own expression and that of putP, a proline transporter (). Therefore, the increased levels of PutA in the pigX mutant strain might suggest a drop in intracellular proline. However, it has been observed that proline transport mutants of Streptomyces coelicolor A3(2) can also lose the ability to degrade proline while still retaining proline biosynthesis. The result was an increase in the production of undecylprodigiosin, which was suggested to be acting as a “metabolic sink” for excess proline (). Finally, PepN, an intracellular aminopeptidase, was increased in the pigX mutant background. In E. coli, PepN has been implicated as the major aminopeptidase involved in the degradation of cytosolic proteins and may have a role in some cellular stress responses (). Therefore, although we have speculated about links between specific altered proteins involved in amino acid metabolism and enhanced Pig production in the pigX mutant, it is possible that PigX plays a more general role in the regulation of amino acid metabolism.
Alanine substitution mutants were constructed in the ELI motif, resulting in two plasmids with altered forms of PigX. A plasmid with an E424A (ELI→ALI) substitution mutation could partially repress Pig production in a pigX mutant strain, demonstrating that the function of PigX was impaired (Fig. ). An E424A L425A I426A (ELI→AAA) triple amino acid substitution mutant plasmid could not complement the pigX mutant for the biosynthesis of Pig (Fig. ). These complementation experiments suggested that the ELI motif was essential for the activity of PigX. To further examine the roles of the different PigX domains, a series of plasmids was constructed encoding different domains of PigX (Fig. ). Strikingly, the EAL domain alone fully complemented Pig production in the pigX mutant strain back to WT levels (Fig. ). Furthermore, plasmids expressing the GGDEF domain alone or in combination with the potential membrane-spanning region had no significant effect on Pig production in the pigX mutant. In addition, the expression of a construct possessing both GGDEF and EAL domains, but not the membrane spanning regions, failed to complement the pigX mutant.
Intracellular Distribution of Iron in Cells Supplied with 59Fe-Chrysobactin—Previous studies have shown that the ferric chrysobactin complex is dissociated inside the cell through a rapid reductive process, making iron available for metabolic needs (). Thus, we used 59Fe-chrysobactin to try to identify intracellular protein targets of iron in E. chrysanthemi cells. We used a double mutant strain deficient in biosynthesis of both chrysobactin and achromobactin (PPV20) to avoid iron exchange between both ligands. In the low iron minimal Tris medium, the doubling time of this mutant was 160 min. The addition of ferric chrysobactin to the medium stimulated the growth rate, the doubling time being 100 min. Cytosolic protein extracts were analyzed by PAGE on nondenaturing gels, and protein-bound iron was visualized by autoradiography as described under “Experimental Procedures.” We first tested a concentration range of 0.06-1 μ 59Fe-chrysobactin added over a period of 40 min (). We detected a continuum of bands increasing in intensity with higher 59Fe concentrations, which could correspond to the banding region revealed by Coomassie Blue staining. In addition, two strong 59Fe signals (designated as 1 and 2 on ), a slowly migrating protein species and a quickly migrating protein doublet, were apparent. A third signal (designated as 3 on ) became visible with concentrations of 59Fe higher than 0.125 μ. We then performed a time course experiment. Protein-bound iron was probed at 10-min intervals over a 40-min period after adding 0.25 μ 59Fe-chrysobactin (). We observed the same 59Fe signals increasing in intensity with time. Thus, iron binds a wide variety of proteins. Those referred to as signals 1 and 3 could be ferritin-like compounds.