Identification of a Third Mn(II) Oxidase Enzyme in Pseudomonas putida GB-1.

UNLABELLED: The oxidation of soluble Mn(II) to insoluble Mn(IV) is a widespread bacterial activity found in a diverse array of microbes. In the Mn(II)-oxidizing bacterium Pseudomonas putida GB-1, two Mn(II) oxidase genes, named mnxG and mcoA, were previously identified; each encodes a multicopper oxidase (MCO)-type enzyme. Expression of these two genes is positively regulated by the response regulator MnxR. Preliminary investigation into putative additional regulatory pathways suggested that the flagellar regulators FleN and FleQ also regulate Mn(II) oxidase activity; however, it also revealed the presence of a third, previously uncharacterized Mn(II) oxidase activity in P. putida GB-1. A strain from which both of the Mn(II) oxidase genes and fleQ were deleted exhibited low levels of Mn(II) oxidase activity. The enzyme responsible was genetically and biochemically identified as an animal heme peroxidase (AHP) with domain and sequence similarity to the previously identified Mn(II) oxidase MopA. In the ΔfleQ strain, P. putida GB-1 MopA is overexpressed and secreted from the cell, where it actively oxidizes Mn. Thus, deletion of fleQ unmasked a third Mn(II) oxidase activity in this strain. These results provide an example of an Mn(II)-oxidizing bacterium utilizing both MCO and AHP enzymes. IMPORTANCE: The identity of the Mn(II) oxidase enzyme in Pseudomonas putida GB-1 has been a long-standing question in the field of bacterial Mn(II) oxidation. In the current work, we demonstrate that P. putida GB-1 employs both the multicopper oxidase- and animal heme peroxidase-mediated pathways for the oxidation of Mn(II), rendering this model organism relevant to the study of both types of Mn(II) oxidase enzymes. The presence of three oxidase enzymes in P. putida GB-1 deepens the mystery of why microorganisms oxidize Mn(II) while providing the field with the tools necessary to address this question. The initial identification of MopA as a Mn(II) oxidase in this strain required the deletion of FleQ, a regulator involved in both flagellum synthesis and biofilm synthesis in Pseudomonas aeruginosa Therefore, these results are also an important step toward understanding the regulation of Mn(II) oxidation.

Elimination of manganese(II,III) oxidation in Pseudomonas putida GB-1 by a double knockout of two putative multicopper oxidase genes.

Bacterial manganese(II) oxidation impacts the redox cycling of Mn, other elements, and compounds in the environment; therefore, it is important to understand the mechanisms of and enzymes responsible for Mn(II) oxidation. In several Mn(II)-oxidizing organisms, the identified Mn(II) oxidase belongs to either the multicopper oxidase (MCO) or the heme peroxidase family of proteins. However, the identity of the oxidase in Pseudomonas putida GB-1 has long remained unknown. To identify the P. putida GB-1 oxidase, we searched its genome and found several homologues of known or suspected Mn(II) oxidase-encoding genes (mnxG, mofA, moxA, and mopA). To narrow this list, we assumed that the Mn(II) oxidase gene would be conserved among Mn(II)-oxidizing pseudomonads but not in nonoxidizers and performed a genome comparison to 11 Pseudomonas species. We further assumed that the oxidase gene would be regulated by MnxR, a transcription factor required for Mn(II) oxidation. Two loci met all these criteria: PputGB1_2447, which encodes an MCO homologous to MnxG, and PputGB1_2665, which encodes an MCO with very low homology to MofA. In-frame deletions of each locus resulted in strains that retained some ability to oxidize Mn(II) or Mn(III); loss of oxidation was attained only upon deletion of both genes. These results suggest that PputGB1_2447 and PputGB1_2665 encode two MCOs that are independently capable of oxidizing both Mn(II) and Mn(III). The purpose of this redundancy is unclear; however, differences in oxidation phenotype for the single mutants suggest specialization in function for the two enzymes.

The molecular biogeochemistry of manganese(II) oxidation.

Micro-organisms capable of oxidizing the redox-active transition metal manganese play an important role in the biogeochemical cycle of manganese. In the present mini-review, we focus specifically on Mn(II)-oxidizing bacteria. The mechanisms by which bacteria oxidize Mn(II) include a two-electron oxidation reaction catalysed by a novel multicopper oxidase that produces Mn(IV) oxides as the primary product. Bacteria also produce organic ligands, such as siderophores, that bind to and stabilize Mn(III). The realization that this stabilized Mn(III) is present in many environments and can affect the redox cycles of other elements such as sulfur has made it clear that manganese and the bacteria that oxidize it profoundly affect the Earth's biogeochemistry.

Mn(II) oxidation in Pseudomonas putida GB-1 is influenced by flagella synthesis and surface substrate.

Bacterially mediated manganese(II) oxidation greatly affects the biogeochemical cycling of Mn and other elements. One species of bacteria that are capable of Mn(II) oxidation is the gamma-proteobacterium Pseudomonas putida GB-1. In this organism, Mn(II) oxidation begins in stationary phase on the outer surface of the cell, forming a layer of insoluble Mn(III,IV) oxides. A random transposon mutagenesis screen isolated 12 mutant strains of P. putida GB-1 that exhibited increased Mn(II) oxidation on solid media relative to wild type. In 8 out of the 12 strains, the transposon had inserted into a putative flagellar gene. Those 8 strains each had motility defects, thus the disrupted genes are part of the P. putida GB-1 flagellar regulon. The flagellar genes identified include putative structural components (FliC, FliD, FlgE, and FlgL) and regulatory proteins (FlgM and FleN). Deletion of either the FleN gene (fleN) or the overlapping gene fliA resulted in increased Mn(II) oxidation, while in-frame deletion of fliF, which encodes an essential component of the basal body, did not. In liquid media, the flagellar mutants exhibited delayed Mn(II) oxidation relative to wild type. These results suggest that bacterial Mn(II) oxidation is regulated in part by flagellar-mediated responses to the surface substrate.

Identification of a two-component regulatory pathway essential for Mn(II) oxidation in Pseudomonas putida GB-1.

Bacterial manganese(II) oxidation has a profound impact on the biogeochemical cycling of Mn and the availability of the trace metals adsorbed to the surfaces of solid Mn(III, IV) oxides. The Mn(II) oxidase enzyme was tentatively identified in Pseudomonas putida GB-1 via transposon mutagenesis: the mutant strain GB-1-007, which fails to oxidize Mn(II), harbors a transposon insertion in the gene cumA. cumA encodes a putative multicopper oxidase (MCO), a class of enzymes implicated in Mn(II) oxidation in other bacterial species. However, we show here that an in-frame deletion of cumA did not affect Mn(II) oxidation. Through complementation analysis of the oxidation defect in GB-1-007 with a cosmid library and subsequent sequencing of candidate genes we show the causative mutation to be a frameshift within the mnxS1 gene that encodes a putative sensor histidine kinase. The frameshift mutation results in a truncated protein lacking the kinase domain. Multicopy expression of mnxS1 restored Mn(II) oxidation to GB-1-007 and in-frame deletion of mnxS1 resulted in a loss of oxidation in the wild-type strain. These results clearly demonstrated that the oxidation defect of GB-1-007 is due to disruption of mnxS1, not cumA::Tn5, and that CumA is not the Mn(II) oxidase. mnxS1 is located upstream of a second sensor histidine kinase gene, mnxS2, and a response regulator gene, mnxR. In-frame deletions of each of these genes also led to the loss of Mn(II) oxidation. Therefore, we conclude that the MnxS1/MnxS2/MnxR two-component regulatory pathway is essential for Mn(II) oxidation in P. putida GB-1.

The hybrid sensor kinase RscS integrates positive and negative signals to modulate biofilm formation in Vibrio fischeri.

Overexpression of the Vibrio fischeri sensor kinase RscS induces expression of the syp (symbiosis polysaccharide) gene cluster and promotes biofilm phenotypes such as wrinkled colony morphology, pellicle formation, and surface adherence. RscS is predicted to be a hybrid sensor kinase with a histidine kinase/ATPase (HATPase) domain, a receiver (Rec) domain, and a histidine phosphotransferase (Hpt) domain. Bioinformatic analysis also revealed the following three potential signal detection domains within RscS: two transmembrane helices forming a transmembrane region (TMR), a large periplasmic (PP) domain, and a cytoplasmic PAS domain. In this work, we genetically dissected the contributions of these domains to RscS function. Substitutions within the carboxy-terminal domain supported identification of RscS as a hybrid sensor kinase; disruption of both the HATPase and Rec domains eliminated induction of syp transcription, wrinkled colony morphology, pellicle formation, and surface adherence, while disruption of Hpt resulted in decreased activity. The PAS domain was also critical for RscS activity; substitutions in PAS resulted in a loss of activity. Generation of a cytoplasmic, N-terminal deletion derivative of RscS resulted in a partial loss of activity, suggesting a role for localization to the membrane and/or sequences within the TMR and PP domain. Finally, substitutions within the first transmembrane helix of the TMR and deletions within the PP domain both resulted in increased activity. Thus, RscS integrates both inhibitory and stimulatory signals from the environment to regulate biofilm formation by V. fischeri.

Multiple factors contribute to keeping levels of the symbiosis regulator RscS low.

Increased activity alleles (rscS1 and rscS2) of the symbiosis regulator RscS induced both syp transcription and biofilm formation in Vibrio fischeri. Neither allele encodes a protein variant; instead they carry mutations near the putative ribosome-binding site and, in the case of rscS1, an additional silent mutation at codon Leu25. In this study, we found that endogenous levels of RscS are very low under the culture conditions examined and that the increased activity alleles dramatically increased the levels of protein. Of the two mutations present in rscS1, the Leu25 mutation, which replaces a rare with a more common Leu codon, appeared to make the greater contribution to increased activity. Our results suggest that RscS levels are maintained at low levels in the cell by the presence of a weak promoter, possible inefficient ribosome binding and the presence of rare codons in the 5' end of the gene. Restriction of RscS levels may be important to prevent spurious signalling by this sensor kinase in the absence of a squid host.

Oxidative Formation and Removal of Complexed Mn(III) by Pseudomonas Species.

The observation of significant concentrations of soluble Mn(III) complexes in oxic, suboxic, and some anoxic waters has triggered a re-evaluation of the previous Mn paradigm which focused on the cycling between soluble Mn(II) and insoluble Mn(III,IV) species as operationally defined by filtration. Though Mn(II) oxidation in aquatic environments is primarily bacterially-mediated, little is known about the effect of Mn(III)-binding ligands on Mn(II) oxidation nor on the formation and removal of Mn(III). Pseudomonas putida GB-1 is one of the most extensively investigated of all Mn(II) oxidizing bacteria, encoding genes for three Mn oxidases (McoA, MnxG, and MopA). P. putida GB-1 and associated Mn oxidase mutants were tested alongside environmental isolates Pseudomonas hunanensis GSL-007 and Pseudomonas sp. GSL-010 for their ability to both directly oxidize weakly and strongly bound Mn(III), and to form these complexes through the oxidation of Mn(II). Using Mn(III)-citrate (weak complex) and Mn(III)-DFOB (strong complex), it was observed that P. putida GB-1, P. hunanensis GSL-007 and Pseudomonas sp. GSL-010 and mutants expressing only MnxG and McoA were able to directly oxidize both species at varying levels; however, no oxidation was detected in cultures of a P. putida mutant expressing only MopA. During cultivation in the presence of Mn(II) and citrate or DFOB, P. putida GB-1, P. hunanensis GSL-007 and Pseudomonas sp. GSL-010 formed Mn(III) complexes transiently as an intermediate before forming Mn(III/IV) oxides with the overall rates and extents of Mn(III,IV) oxide formation being greater for Mn(III)-citrate than for Mn(III)-DFOB. These data highlight the role of bacteria in the oxidative portion of the Mn cycle and suggest that the oxidation of strong Mn(III) complexes can occur through enzymatic mechanisms involving multicopper oxidases. The results support the observations from field studies and further emphasize the complexity of the geochemical cycling of manganese.

A hydrophobic patch on the flap-tip helix of E.coli RNA polymerase mediates sigma(70) region 4 function.

The Escherichia coli RNA polymerase beta subunit contains a flexible flap domain that interacts with region 4 of sigma(70) to position it for recognition of the -35 element of promoters. We report that this function depends on a hydrophobic patch on one face of the short stretch of alpha helix located at the tip of the flap domain, called the flap-tip helix. Disruption of the hydrophobic patch by the substitution of hydrophilic or charged amino acids resulted in a loss of the interaction between the flap and sigma region 4, as determined by protease sensitivity assays, and impaired transcription from -35-dependent promoters. We suggest that contact of the flap-tip helix hydrophobic patch to the sigma region 4 hydrophobic core is essential for stable interaction of the flap-tip helix with region 4. This contact allowed region 4.2 recognition of the -35 promoter element and appeared to stabilize region 4 interaction with the beta' Zn(2+) binding domain. Our studies failed to detect any role for sigma region 1.1 in establishing or maintaining the flap-sigma region 4 interaction, consistent with recent reports placing sigma region 1.1 in the downstream DNA channel.

Effects of exogenous pyoverdines on Fe availability and their impacts on Mn(II) oxidation by Pseudomonas putida GB-1.

Pseudomonas putida GB-1 is a Mn(II)-oxidizing bacterium that produces pyoverdine-type siderophores (PVDs), which facilitate the uptake of Fe(III) but also influence MnO2 formation. Recently, a non-ribosomal peptide synthetase mutant that does not synthesize PVD was described. Here we identified a gene encoding the PVDGB-1 (PVD produced by strain GB-1) uptake receptor (PputGB1_4082) of strain GB-1 and confirmed its function by in-frame mutagenesis. Growth and other physiological responses of these two mutants and of wild type were compared during cultivation in the presence of three chemically distinct sets of PVDs (siderotypes n°1, n°2, and n°4) derived from various pseudomonads. Under iron-limiting conditions, Fe(III) complexes of various siderotype n°1 PVDs (including PVDGB-1) allowed growth of wild type and the synthetase mutant, but not the receptor mutant, confirming that iron uptake with any tested siderotype n°1 PVD depended on PputGB1_4082. Fe(III) complexes of a siderotype n°2 PVD were not utilized by any strain and strongly induced PVD synthesis. In contrast, Fe(III) complexes of siderotype n°4 PVDs promoted the growth of all three strains and did not induce PVD synthesis by the wild type, implying these complexes were utilized for iron uptake independent of PputGB1_4082. These differing properties of the three PVD types provided a way to differentiate between effects on MnO2 formation that resulted from iron limitation and others that required participation of the PVDGB-1 receptor. Specifically, MnO2 production was inhibited by siderotype n°1 but not n°4 PVDs indicating PVD synthesis or PputGB1_4082 involvement rather than iron-limitation caused the inhibition. In contrast, iron limitation was sufficient to explain the inhibition of Mn(II) oxidation by siderotype n°2 PVDs. Collectively, our results provide insight into how competition for iron via siderophores influences growth, iron nutrition and MnO2 formation in more complex environmental systems.

Pyoverdine synthesis by the Mn(II)-oxidizing bacterium Pseudomonas putida GB-1.

When iron-starved, the Mn(II)-oxidizing bacteria Pseudomonas putida strains GB-1 and MnB1 produce pyoverdines (PVDGB-1 and PVDMnB1), siderophores that both influence iron uptake and inhibit manganese(II) oxidation by these strains. To explore the properties and genetics of a PVD that can affect manganese oxidation, LC-MS/MS, and various siderotyping techniques were used to identify the peptides of PVDGB-1 and PVDMnB1 as being (for both PVDs): chromophore-Asp-Lys-OHAsp-Ser-Gly-aThr-Lys-cOHOrn, resembling a structure previously reported for P. putida CFML 90-51, which does not oxidize Mn. All three strains also produced an azotobactin and a sulfonated PVD, each with the peptide sequence above, but with unknown regulatory or metabolic effects. Bioinformatic analysis of the sequenced genome of P. putida GB-1 suggested that a particular non-ribosomal peptide synthetase (NRPS), coded by the operon PputGB1_4083-4086, could produce the peptide backbone of PVDGB-1. To verify this prediction, plasmid integration disruption of PputGB1_4083 was performed and the resulting mutant failed to produce detectable PVD. In silico analysis of the modules in PputGB1_4083-4086 predicted a peptide sequence of Asp-Lys-Asp-Ser-Ala-Thr-Lsy-Orn, which closely matches the peptide determined by MS/MS. To extend these studies to other organisms, various Mn(II)-oxidizing and non-oxidizing isolates of P. putida, P. fluorescens, P. marincola, P. fluorescens-syringae group, P. mendocina-resinovorans group, and P. stutzerii group were screened for PVD synthesis. The PVD producers (12 out of 16 tested strains) were siderotyped and placed into four sets of differing PVD structures, some corresponding to previously characterized PVDs and some to novel PVDs. These results combined with previous studies suggested that the presence of OHAsp or the flexibility of the pyoverdine polypeptide may enable efficient binding of Mn(III).

The sugar phosphotransferase system of Vibrio fischeri inhibits both motility and bioluminescence.

Magnesium-dependent induction of Vibrio fischeri flagellar (Mif) biogenesis depends upon two diguanylate cyclases, suggesting an inhibitory role for cyclic di-GMP. Here, we report that cells defective for the sugar phosphotransferase system (PTS) exhibited a magnesium-independent phenotype similar to that of mutants of the Mif pathway. Unlike Mif mutants, PTS mutants also were hyperbioluminescent.

The symbiosis regulator rscS controls the syp gene locus, biofilm formation and symbiotic aggregation by Vibrio fischeri.

Successful colonization of a eukaryotic host by a microbe involves complex microbe-microbe and microbe-host interactions. Previously, we identified in Vibrio fischeri a putative sensor kinase, RscS, required for initiating symbiotic colonization of its squid host Euprymna scolopes. Here, we analysed the role of rscS by isolating an allele, rscS1, with increased activity. Multicopy rscS1 activated transcription of genes within the recently identified symbiosis polysaccharide (syp) cluster. Wild-type cells carrying rscS1 induced aggregation phenotypes in culture, including the formation of pellicles and wrinkled colonies, in a syp-dependent manner. Colonies formed by rscSl-expressing cells produced a matrix not found in control colonies and largely lost in an rscSl-expressing sypN mutant. Finally, multicopy rscS1 provided a colonization advantage over control cells and substantially enhanced the ability of wild-type cells to aggregate on the surface of the symbiotic organ of E. scolopes; this latter phenotype similarly depended upon an intact syp locus. These results suggest that transcription induced by RscS-mediated signal transduction plays, a key role in colonization at the aggregation stage by modifying the cell surface and increasing the ability of the cells to adhere to one another and/or to squid-secreted mucus.

Diguanylate cyclases control magnesium-dependent motility of Vibrio fischeri.

Flagellar biogenesis and hence motility of Vibrio fischeri depends upon the presence of magnesium. In the absence of magnesium, cells contain few or no flagella and are poorly motile or nonmotile. To dissect the mechanism by which this regulation occurs, we screened transposon insertion mutants for those that could migrate through soft agar medium lacking added magnesium. We identified mutants with insertions in two distinct genes, VF0989 and VFA0959, which we termed mifA and mifB, respectively, for magnesium-dependent induction of flagellation. Each gene encodes a predicted membrane-associated protein with diguanylate cyclase activity. Consistent with that activity, introduction into V. fischeri of medium-copy plasmids carrying these genes inhibited motility. Furthermore, multicopy expression of mifA induced other phenotypes known to be correlated with diguanylate cyclase activity, including cellulose biosynthesis and biofilm formation. To directly test their function, we introduced the wild-type genes on high-copy plasmids into Escherichia coli. We assayed for the production of cyclic di-GMP using two-dimensional thin-layer chromatography and found that strains carrying these plasmids produced a small but reproducible spot that migrated with an R(f) value consistent with cyclic di-GMP that was not produced by strains carrying the vector control. Disruptions of mifA or mifB increased flagellin levels, while multicopy expression decreased them. Semiquantitative reverse transcription-PCR experiments revealed no significant difference in the amount of flagellin transcripts produced in either the presence or absence of Mg(2+) by either vector control or mifA-overexpressing cells, indicating that the impact of magnesium and cyclic-di-GMP primarily acts following transcription. Finally, we present a model for the roles of magnesium and cyclic di-GMP in the control of motility of V. fischeri.