Biosynthesis and Bioactivity of Prodiginine Analogs in Marine Bacteria, Pseudoalteromonas: A Mini Review.

The Prodiginine family consists of primarily red-pigmented tripyrrole secondary metabolites that were first characterized in the Gram-negative bacterial species Serratia marcescens and demonstrates a wide array of biological activities and applications. Derivatives of prodiginine have since been characterized in the marine γ-proteobacterium, Pseudoalteromonas. Although biosynthetic gene clusters involved in prodiginine synthesis display homology among genera, there is an evident structural difference in the resulting metabolites. This review will summarize prodiginine biosynthesis, bioactivity, and gene regulation in Pseudoalteromonas in comparison to the previously characterized species of Serratia, discuss the ecological contributions of Pseudoalteromonas in the marine microbiome and their eukaryotic hosts, and consider the importance of modern functional genomics and classic DNA manipulation to understand the overall prodiginine biosynthesis pathway.

Draft Genome Sequence of Marine Sponge Symbiont Pseudoalteromonas luteoviolacea IPB1, Isolated from Hilo, Hawaii.

We report here the 6.0-Mb draft genome assembly of Pseudoalteromonas luteoviolacea strain IPB1 that was isolated from the Hawaiian marine sponge Iotrochota protea Genome mining complemented with bioassay studies will elucidate secondary metabolite biosynthetic pathways and will help explain the ecological interaction between host sponge and microorganism.

Roles of siderophores, oxalate, and ascorbate in mobilization of iron from hematite by the aerobic bacterium Pseudomonas mendocina.

In aerobic, circumneutral environments, the essential element Fe occurs primarily in scarcely soluble mineral forms. We examined the independent and combined effects of a siderophore, a reductant (ascorbate), and a low-molecular-weight carboxylic acid (oxalate) on acquisition of Fe from the mineral hematite (alpha-Fe(2)O(3)) by the obligate aerobe Pseudomonas mendocina ymp. A site-directed DeltapmhA mutant that was not capable of producing functional siderophores (i.e., siderophore(-) phenotype) did not grow on hematite as the only Fe source. The concentration of an added exogenous siderophore (1 microM desferrioxamine B [DFO-B]) needed to restore wild-type (WT)-like growth kinetics to the siderophore(-) strain was approximately 50-fold less than the concentration of the siderophore secreted by the WT organism grown under the same conditions. The roles of a reductant (ascorbate) and a simple carboxylic acid (oxalate) in the Fe acquisition process were examined in the presence and absence of the siderophore. Addition of ascorbate (50 microM) alone restored the growth of the siderophore(-) culture to the WT levels. A higher concentration of oxalate (100 microM) had little effect on the growth of a siderophore(-) culture; however, addition of 0.1 muM DFO-B and 100 muM oxalate restored the growth of the mutant to WT levels when the oxalate was prereacted with the hematite, demonstrating that a metabolizing culture benefits from a synergistic effect of DFO-B and oxalate.

Pyruvate carboxylase is involved in metabolism of mimosine by Rhizobium sp. strain TAL1145.

The objective of this study was to determine the role of midK, which encodes a protein similar to pyruvate carboxylase, in mimosine degradation by Rhizobium sp. strain TAL1145. The midK gene is located downstream of midR in the cluster of genes for mimosine degradation in Rhizobium sp. strain TAL1145. The midK mutants of TAL1145 degraded mimosine slower than the wild-type. These mutants could utilize pyruvate as a source of carbon, indicating that there is another pyruvate carboxylase (pyc) gene in TAL1145. Two classes of clones were isolated from the library of TAL1145 by complementing a pyc mutant of Rhizobium etli, one class contained midK, while the other carried pyc. Both midK and pyc of TAL1145 complemented the midK mutant for mimosine degradation, and also the R. etli pyc mutant for pyruvate utilization. The midK-encoded pyruvate carboxylase was required for an efficient conversion of mimosine into 3-hydroxy-4-pyridone (HP).

The cloned 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene from Sinorhizobium sp. strain BL3 in Rhizobium sp. strain TAL1145 promotes nodulation and growth of Leucaena leucocephala.

The objective of this study was to determine the role of 1-aminocyclopropane-1-carboxylate (ACC) deaminase of symbionts in nodulation and growth of Leucaena leucocephala. The acdS genes encoding ACC deaminase were cloned from Rhizobium sp. strain TAL1145 and Sinorhizobium sp. BL3 in multicopy plasmids, and transferred to TAL1145. The BL3-acdS gene greatly enhanced ACC deaminase activity in TAL1145 compared to the native acdS gene. The transconjugants of TAL1145 containing the native or BL3 acdS gene could grow in minimal media containing 1.5mM ACC, whereas BL3 could tolerate up to 3mM ACC. The TAL1145 acdS gene was inducible by mimosine and not by ACC, while the BL3 acdS gene was highly inducible by ACC and not by mimosine. The transconjugants of TAL1145 containing the native- and BL3-acdS genes formed nodules with greater number and sizes, and produced higher root mass on L. leucocephala than by TAL1145. This study shows that the introduction of multiple copies of the acdS gene increased ACC deaminase activities of TAL1145 and enhanced its symbiotic efficiency on L. leucocephala.

Identification, isolation, and analysis of a gene cluster involved in iron acquisition by Pseudomonas mendocina ymp.

Microbial acquisition of iron from natural sources in aerobic environments is a little-studied process that may lead to mineral instability and trace metal mobilization. Pseudomonas mendocina ymp was isolated from the Yucca Mountain Site for long-term nuclear waste storage. Its ability to solubilize a variety of Fe-containing minerals under aerobic conditions has been previously investigated but its molecular and genetic potential remained uncharacterized. Here, we have shown that the organism produces a hydroxamate and not a catecholate-based siderophore that is synthesized via non-ribosomal peptide synthetases. Gene clustering patterns observed in other Pseudomonads suggested that hybridizing multiple probes to the same library could allow for the identification of one or more clusters of syntenic siderophore-associated genes. Using this approach, two independent clusters were identified. An unfinished draft genome sequence of P. mendocina ymp indicated that these mapped to two independent contigs. The sequenced clusters were investigated informatically and shown to contain respectively a potentially complete set of genes responsible for siderophore biosynthesis, uptake, and regulation, and an incomplete set of genes with low individual homology to siderophore-associated genes. A mutation in the cluster's pvdA homolog (pmhA) resulted in a siderophore-null phenotype, which could be reversed by complementation. The organism likely produces one siderophore with possibly different isoforms and a peptide backbone structure containing seven residues (predicted sequence: Acyl-Asp-Dab-Ser-fOHOrn-Ser-fOHorn). A similar approach could be applied for discovery of Fe- and siderophore-associated genes in unsequenced or poorly annotated organisms.

The pydA-pydB fusion gene produces an active dioxygenase-hydrolase that degrades 3-hydroxy-4-pyridone, an intermediate of mimosine metabolism.

The objective of this research was to construct a pydA-pydB hybrid gene that encodes a functional dioxygenase-hydrolase (PydA-PydB) fusion protein for degradation of 3-hydroxy-4-pyridone (HP). HP is an intermediate in both synthesis and degradation of mimosine, a toxic amino acid produced by the tree legume Leucaena leucocephala. Computer-generated models of the fusion proteins suggested that joining of PydA and PydB with 0, 3, or 7 glycine residues as a linker should produce a functional PydA-PydB fusion protein. Accordingly, three hybrid genes, G0, G3, and G7, were constructed in which pydA and pydB were connected with 0, 9, and 21 nucleotides, respectively, encoding the glycine residues of the linker region. When these hybrid genes were expressed in Rhizobium and Escherichia coli, only one of them, G3, produced a functional PydA-PydB fusion protein, having both the dioxygenase and hydrolase activities. The G3 hybrid gene could complement both pydA and pydB mutants of Rhizobium, and E. coli lysate containing the overexpressed G3 protein was able to degrade HP. This hybrid gene may be useful for developing mimosine-free L. leucocephala plants in the future.

pyd genes of Rhizobium sp. strain TAL1145 are required for degradation of 3-hydroxy-4-pyridone, an aromatic intermediate in mimosine metabolism.

Rhizobium sp. strain TAL1145 degrades the Leucaena toxin mimosine and its degradation product 3-hydroxy-4-pyridone (HP). The aim of this investigation is to characterize the Rhizobium genes for HP degradation and transport. These genes were localized by subcloning and mutagenesis on a previously isolated cosmid, pUHR263, containing mid genes of TAL1145 required for mimosine degradation. Two structural genes, pydA and pydB, encoding a metacleavage dioxygenase and a hydrolase, respectively, are required for degradation of HP, and three genes, pydC, pydD, and pydE, encoding proteins of an ABC transporter, are involved in the uptake of HP by TAL1145. These genes are induced by HP, although both pydA and pydB show low levels of expression without HP. pydA and pydB are cotranscribed, while pydC, pydD, and pydE are each transcribed from separate promoters. PydA and PydB show no homology with other dioxygenases and hydrolases in Sinorhizobium meliloti, Mesorhizobium loti, and Bradyrhizobium japonicum. Among various root nodule bacteria, the ability to degrade mimosine or HP is unique to some Leucaena-nodulating Rhizobium strains.

Remediation of benzo[a]pyrene and chrysene-contaminated soil with industrial hemp (Cannabis sativa).

The phytoremediation, with industrial hemp (Cannabis sativa), of a Hawaiian silty clay soil contaminated with two polycyclic aromatic hydrocarbons (PAHs), chrysene and benzo[a]pyrene, was studied. Hemp showed a very high tolerance to the contaminants. The growth rates of hemp, compared with control, in soils fortified with chrysene and benzo[a]pyrene at concentrations of each varying from 25 to 200 micrograms/g were consistently above 100%. The plants grew from seed for 45 days in soil fortified with PAHs at concentrations of 25, 50, and 75 micrograms/g. Controls were pots with contaminated soil but no plant. PAHs levels were significantly reduced in all pots (control and seeded pots), expect for one set at a high concentration of chrysene, which may be due to uneven spiking. A time course study over 28 days was done to monitor changes of microbial count and levels of chrysene. Little changes were observed for the total microbial count in the soil, and the concentration of chrysene in the soil decreased slightly in the pots containing plants. However, the chrysene levels in those pots were consistently lower than those in the pots without plants.