Morphology engineering for enhanced production of medium-chain-length polyhydroxyalkanoates in Pseudomonas mendocina NK-01.

Polyhydroxyalkanoates (PHAs) can be produced by microorganisms from renewable resources and are regarded as promising bioplastics to replace petroleum-based plastics. A medium-chain-length PHAs (mcl-PHA)-producing strain Pseudomonas mendocina NK-01 was isolated previously by our lab and its whole-genome sequence is currently available. Morphology engineering of manipulating cell morphology-related genes has been applied for enhanced accumulation of the intracellular biopolymer short-chain-length PHAs (scl-PHA). However, it has not yet been reported to improve the yield of mcl-PHA by morphology engineering so far. In this work, several well-characterized cell morphology-related genes, including the cell fission ring (Z-ring) location genes minCD, peptidoglycan degradation gene nlpD, actin-like cytoskeleton protein gene mreB, Z-ring formation gene ftsZ, and FtsZ inhibitor gene sulA, were intensively investigated for their impacts on the cell morphology and mcl-PHA accumulation by gene knockout and overexpression in P. mendocina NKU, a upp knockout mutant of P. mendocina NK-01. For a minCD knockout mutant P. mendocina NKU-∆minCD, the average cell length was obviously increased and the mcl-PHA production was improved. However, the nlpD knockout mutant had a shorter cell length and lower mcl-PHA yield compared with P. mendocina NKU. Overexpression of mreB in P. mendocina NKU resulted in spherical cells. When ftsZ was overexpressed in P. mendocina NKU, the cell division was accelerated and the mcl-PHA titer was improved. Furthermore, mreB, ftsZ, or sulA was overexpressed in P. mendocina NKU-∆minCD. Consequently, the mcl-PHA titers were all increased compared with P. mendocina NKU-∆minCD carrying the empty vector. The multiple fission pattern was finally achieved in ftsZ-overexpressing NKU-∆minCD. In this work, improved production of mcl-PHA in P. mendocina NK-01 has been achieved by morphology engineering. This work provides an alternative strategy to enhance mcl-PHA accumulation in mcl-PHA-producing strains.

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.

Identification of a denitrifying bacterium and verification of its anaerobic ammonium oxidation ability.

A strain D3 of denitrifying bacterium was isolated from an anammox reactor, and identified as Pseudomonas mendocina based on the morphological and physiological assay, Vitek test, Biolog test, (G + C) mol% content, and 16S rDNA phylogenetic analysis. As a typical denitrifying bacterium, strain D3 achieved the maximal nitrate reduction rate of 26.2 mg/(L x d) at the nitrate concentration of 88.5 mg N/L. The optimal pH and growth temperature were 7.84 and 34.9 degrees C, respectively. Strain D3 was able to oxidize ammonia under anaerobic condition. The maximum nitrate and ammonium utilization rates were 6.37 mg/(L x d) and 3.34 mg/(L x d), respectively, and the consumption ratio of ammonia to nitrate was 1:1.91. Electron microscopic observation revealed peculiar cell inclusions in strain D3. Because of its relation to anammox activity, strain D3 was presumed to be anammoxosome. The present investigation proved that denitrifying bacteria have the anammox ability, and the results have engorged the range of anammox populations.