Molecular weight-dependent degradation of D-lactate-containing polyesters by polyhydroxyalkanoate depolymerases from Variovorax sp. C34 and Alcaligenes faecalis T1.

Polyhydroxyalkanoate depolymerase derived from Variovorax sp. C34 (PhaZVs) was identified as the first enzyme that is capable of degrading isotactic P[67 mol% (R)-lactate(LA)-co-(R)-3-hydroxybutyrate(3HB)] [P(D-LA-co-D-3HB)]. This study aimed at analyzing the monomer sequence specificity of PhaZVs for hydrolyzing P(LA-co-3HB) in comparison with a P(3HB) depolymerase from Alcaligenes faecalis T1 (PhaZAf) that did not degrade the same copolymer. Degradation of P(LA-co-3HB) by action of PhaZVs generated dimers, 3HB-3HB, 3HB-LA, LA-3HB, and LA-LA, and the monomers, suggesting that PhaZVs cleaved the linkages between LA and 3HB units and between LA units. To provide a direct evidence for the hydrolysis of these sequences, the synthetic methyl trimers, 3HB-3HB-3HB, LA-LA-3HB, LA-3HB-LA, and 3HB-LA-LA, were treated with the PhaZs. Unexpectedly, not only PhaZVs but also PhaZAf hydrolyzed all of these substrates, namely PhaZAf also cleaved LA-LA linkage. Considering the fact that both PhaZs did not degrade P[(R)-LA] (PDLA) homopolymer, the cleavage capability of LA-LA linkage by PhaZs was supposed to depend on the length of the LA-clustering region in the polymer chain. To test this hypothesis, PDLA oligomers (6 to 40 mer) were subjected to the PhaZ assay, revealing that there was an inverse relationship between molecular weight of the substrates and their hydrolysis efficiency. Moreover, PhaZVs exhibited the degrading activity toward significantly longer PDLA oligomers compared to PhaZAf. Therefore, the cleaving capability of PhaZs used here toward the D-LA-based polymers containing the LA-clustering region was strongly associated with the substrate length, rather than the monomer sequence specificity of the enzyme.

Cell size and the initiation of DNA replication in bacteria.

In eukaryotes, DNA replication is coupled to the cell cycle through the actions of cyclin-dependent kinases and associated factors. In bacteria, the prevailing view, based primarily from work in Escherichia coli, is that growth-dependent accumulation of the highly conserved initiator, DnaA, triggers initiation. However, the timing of initiation is unchanged in Bacillus subtilis mutants that are ~30% smaller than wild-type cells, indicating that achievement of a particular cell size is not obligatory for initiation. Prompted by this finding, we re-examined the link between cell size and initiation in both E. coli and B. subtilis. Although changes in DNA replication have been shown to alter both E. coli and B. subtilis cell size, the converse (the effect of cell size on DNA replication) has not been explored. Here, we report that the mechanisms responsible for coordinating DNA replication with cell size vary between these two model organisms. In contrast to B. subtilis, small E. coli mutants delayed replication initiation until they achieved the size at which wild-type cells initiate. Modest increases in DnaA alleviated the delay, supporting the view that growth-dependent accumulation of DnaA is the trigger for replication initiation in E. coli. Significantly, although small E. coli and B. subtilis cells both maintained wild-type concentration of DnaA, only the E. coli mutants failed to initiate on time. Thus, rather than the concentration, the total amount of DnaA appears to be more important for initiation timing in E. coli. The difference in behavior of the two bacteria appears to lie in the mechanisms that control the activity of DnaA.

Bacterial Population Changes during the Degradation Process of a Lactate (LA)-Enriched Biodegradable Polymer in River Water: LA-Cluster Preferable Bacterial Consortium.

The lactate-based polyester poly[lactate (LA)-co-3-hydroxybutyrate (3HB)], termed LAHB, is a highly transparent and flexible bio-based polymeric material. There are many unknowns regarding its degradation process in riverine environments, especially the changes in bacterial flora that might result from its degradation and the identities of any LAHB-degrading bacteria. LAHB were immersed in the river water samples (A and B), and LAHB degradation was observed in terms of the weight change of the polymer and the microscopic changes on the polymer surfaces. A metagenomic analysis of microorganisms was conducted to determine the effect of LAHB degradation on the aquatic environment. The bacterial flora obtained from beta diversity analysis differed between the two river samples. The river A water sample showed the simultaneous degradation of LA and 3HB even though the copolymer was LA-enriched, suggesting preferable hydrolysis of the LA-enriched segments. In contrast, only 3HB degraded for the LAHB in the river B water sample. The linear discriminant analysis effect size (LEfSe) analysis revealed 14 bacteria that were significantly increased in the river A water sample during LAHB degradation, suggesting that these bacteria preferentially degraded and assimilated LA-clustering polymers. Our metagenomic analysis provides useful insights into the dynamic changes in microbial communities and LA-clustering polymer-degrading bacteria.

Participation of chromosome segregation protein ParAI of Vibrio cholerae in chromosome replication.

Vibrio cholerae carries homologs of plasmid-borne parA and parB genes on both of its chromosomes. The par genes help to segregate many plasmids and chromosomes. Here we have studied the par genes of V. cholerae chromosome I. Earlier studies suggested that ParBI binds to the centromeric site parSI near the origin of replication (oriI), and parSI-ParBI complexes are placed at the cell poles by ParAI. Deletion of parAI and parSI caused the origin-proximal DNA to be less polar. Here we found that deletion of parBI also resulted in a less polar localization of oriI. However, unlike the deletion of parAI, the deletion of parBI increased the oriI number. Replication was normal when both parAI and parBI were deleted, suggesting that ParBI mediates its action through ParAI. Overexpression of ParAI in a parABI-deleted strain also increased the DNA content. The results are similar to those found for Bacillus subtilis, where ParA (Soj) stimulates replication and this activity is repressed by ParB (SpoOJ). As in B. subtilis, the stimulation of replication most likely involves the replication initiator DnaA. Our results indicate that control of chromosomal DNA replication is an additional function of chromosomal par genes conserved across the Gram-positive/Gram-negative divide.

Enhanced production of lactate-based polyesters in Escherichia coli from a mixture of glucose and xylose by Mlc-mediated catabolite derepression.

Lignocellulose-utilizing biorefinery is a promising strategy for the sustainable production of value-added products such as bio-based polymers. Simultaneous consumption of glucose and xylose in Escherichia coli was achieved by overexpression of the gene encoding Mlc, a multiple regulator of glucose and xylose uptake. This catabolite derepression gave the enhancement in the production of poly (15 mol% lactate-co-3-hydroxybutyrate), up to 65% from 50% (wild-type strain) in the cellular contents, of the Mlc-overexpressing strain of E. coli on a mixture of glucose and xylose as carbon sources. Microscopic analysis indicated that the Mlc-overexpressing strain showed the enlargement of cell volume in the presence and absence of polymer production, consequently making an expanded volumetric space available for enhanced polymer accumulation. The enhanced polymer production by the catabolite derepression was also reproducible using the biomass, Miscanthus×giganteus (hybrid Miscanthus), which was cultivated in the farm of Hokkaido University.

Genome-wide screening of transcription factor deletion targets in Escherichia coli for enhanced production of lactate-based polyesters.

Engineered Escherichia coli is a useful platform for production of lactate (LA)-based polyester poly[LA-co-3-hydroxybutyrate (3HB)] from renewable sugars. Here we screened all non-lethal transcription factor deletions of E. coli for efficient production of the polymer. This approach aimed at drawing out the latent potential of the host for efficient polymer production via indirect positive effects. Among 252 mutants from Keio Collection tested, eight mutants (ΔpdhR, ΔcspG, ΔyneJ, ΔchbR, ΔyiaU, ΔcreB, ΔygfI and ΔnanK) accumulated greater amount of polymer (6.2-10.1 g/L) compared to the parent strain E. coli BW25113 (5.1 g/L). The mutants increased polymer production per cell (1.1-1.5-fold) without significant change in cell density. The yield of the polymer from glucose was also higher for the selected mutants (0.34-0.38 g/g) than the parent strain (0.27 g/g). Therefore, the deletions of transcription factors should channel the carbon flux towards polymer production. It should be noted that the screening employed in this study identified beneficial mutants without analyzing causal relationship between the mutation and the enhanced polymer production. This approach, therefore, should be applicable to broad range of fermentation productions.

Proteome analysis of Pseudomonas putida F1 genes induced in soil environments.

Knowledge of the gene expression dynamics of a single soil bacterial strain contributes to the understanding of its behaviour, physiological state and surrounding microenvironment. Genes expressed in soil environments rather than in laboratory media are considered to particularly relevant. Here, we compared genome-wide gene expression profiles of the bacterium Pseudomonas putida F1 inoculated in three different types of nonsterile soils deduced using proteome analysis via sodium dodecyl sulfate-polyacrylamide gel electrophoresis combined with liquid chromatography-tandem mass spectrometry. Proteins commonly detected in all three samples and involved with bacterial growth and fundamental metabolism were excluded. Nine proteins were identified as specifically expressed in soil including an aldehyde dehydrogenase, a nitric oxide dioxygenase and five proteins encoded by a cluster of metabolism-associated genes. Expression factor analysis revealed that the nitric oxide dioxygenase-coding gene was induced by nitric oxide and the five clustered genes were induced under phosphate starvation. The expression of these genes can be attributed to response to soil environmental stimuli surrounding the F1 cells. These results strongly suggest that our soil metaproteome approach is useful for understanding the autecology and lifestyle of a single bacterial strain in soil environments and allows the prediction of the microenvironment surrounding the bacterial cells.

Enhanced cellular content and lactate fraction of the poly(lactate-co-3-hydroxybutyrate) polyester produced in recombinant Escherichia coli by the deletion of σ factor RpoN.

A new approach at the transcriptional level was applied to lactate-based polyester production. Four σ factor disruptants, ΔrpoN, ΔrpoS, ΔfliA and ΔfecI, of Escherichia coli were used as hosts for poly(lactate-co-3-hydroxybutyrate) production from glucose. Among them, ΔrpoN caused dual positive effects of polymer production, enhanced cellular content and lactate fraction.

Indirect positive effects of a sigma factor RpoN deletion on the lactate-based polymer production in Escherichia coli.

The production of bacterial polyesters, polyhydroxyalkanoates (PHAs), has been improved by several rational approaches such as overexpression and/or engineering of the enzymes directly related to PHA biosynthetic pathways. In this study, a new approach at transcription level has been applied to a new category of the copolymer of lactate (LA) and 3-hydroxybutyrate (3HB), P(LA-co-3HB). When the 4 disrupting mutants of sigma factors in Escherichia coli, rpoN, rpoS, fliA, fecI, were used as platforms for production of P(LA-co-3HB), increases in the production level and LA fraction of the copolymer were observed for the mutant strain with rpoN disruption. These positive impacts on the polymer production were caused in an "indirect manner" via changes in the multiple genes governed by RpoN. A genome-wide engineering by sigma factors would be a versatile approach for the production of value-added products of interest and available for combination with the other beneficial tools.

MtgA Deletion-Triggered Cell Enlargement of Escherichia coli for Enhanced Intracellular Polyester Accumulation.

Bacterial polyester polyhydroxyalkanoates (PHAs) have been produced in engineered Escherichia coli, which turned into an efficient and versatile platform by applying metabolic and enzyme engineering approaches. The present study aimed at drawing out the latent potential of this organism using genome-wide mutagenesis. To meet this goal, a transposon-based mutagenesis was carried out on E. coli, which was transformed to produce poly(lactate-co-3-hydroxybutyrate) from glucose. A high-throughput screening of polymer-accumulating cells on Nile red-containing plates isolated one mutant that produced 1.8-fold higher quantity of polymer without severe disadvantages in the cell growth and monomer composition of the polymer. The transposon was inserted into the locus within the gene encoding MtgA that takes part, as a non-lethal component, in the formation of the peptidoglycan backbone. Accordingly, the mtgA-deleted strain E. coli JW3175, which was a derivate of superior PHA-producing strain BW25113, was examined for polymer production, and exhibited an enhanced accumulation of the polymer (7.0 g/l) compared to the control (5.2 g/l). Interestingly, an enlargement in cell width associated with polymer accumulation was observed in this strain, resulting in a 1.6-fold greater polymer accumulation per cell compared to the control. This result suggests that the increase in volumetric capacity for accumulating intracellular material contributed to the enhanced polymer production. The mtgA deletion should be combined with conventional engineering approaches, and thus, is a promising strategy for improved production of intracellularly accumulated biopolymers.

Protein co-migration database (PCoM -DB) for Arabidopsis thylakoids and Synechocystis cells.

Protein-protein interactions are critical for most cellular processes; however, many remain to be identified. Here, to comprehensively identify protein complexes in photosynthetic organisms, we applied the recently developed approach of blue native PAGE (BN-PAGE) coupled with LC-MS/MS to the thylakoid proteins of Arabidopsis thaliana and the whole cell proteins of whole cell proteins of Synechocystis sp. PCC 6803. We identified 245 proteins from the purified Arabidopsis thylakoid membranes and 1,458 proteins from the whole cells of Synechocystis using the method. Next, we generated protein migration profiles that were assessed by plotting the label-free estimations of protein abundances versus migration distance in BN-PAGE. Comparisons between the migration profiles of the major photosynthetic complexes and their band patterns showed that the protein migration profiles were well correlated. Thus, the protein migration profiles allowed us to estimate the molecular size of each protein complex and to identify co-migrated proteins with the proteins of interest by determining the protein pairs that contained peaks in the same gel slice. Finally, we built the protein co-migration database for photosynthetic organisms (PCoM-DB: http://pcomdb.lowtem.hokudai.ac.jp/proteins/top) to make our data publicly accessible online, which stores the analyzed data with a user-friendly interface to compare the migration profiles of proteins of interest. It helps users to find unidentified protein complexes in Arabidopsis thylakoids and Synechocystis cells. The accumulation of the data from the BN-PAGE coupled with LC-MS/MS should reveal unidentified protein complexes and should aid in understanding the adaptation and the evolution of photosynthetic organisms.

Insensitivity of chromosome I and the cell cycle to blockage of replication and segregation of Vibrio cholerae chromosome II.

UNLABELLED: Vibrio cholerae has two chromosomes (chrI and chrII) whose replication and segregation are under different genetic controls. The region covering the replication origin of chrI resembles that of the Escherichia coli chromosome, and both origins are under control of the highly conserved initiator, DnaA. The origin region of chrII resembles that of plasmids that have iterated initiator-binding sites (iterons) and is under control of the chrII-specific initiator, RctB. Both chrI and chrII encode chromosome-specific orthologs of plasmid partitioning proteins, ParA and ParB. Here, we have interfered with chrII replication, segregation, or both, using extra copies of sites that titrate RctB or ParB. Under these conditions, replication and segregation of chrI remain unaffected for at least 1 cell cycle. In this respect, chrI behaves similarly to the E. coli chromosome when plasmid maintenance is disturbed in the same cell. Apparently, no checkpoint exists to block cell division before the crippled chromosome is lost by a failure to replicate or to segregate. Whether blocking chrI replication can affect chrII replication remains to be tested. IMPORTANCE: Chromosome replication, chromosome segregation, and cell division are the three main events of the cell cycle. They occur in an orderly fashion once per cell cycle. How the sequence of events is controlled is only beginning to be answered in bacteria. The finding of bacteria that possess more than one chromosome raises the important question: how are different chromosomes coordinated in their replication and segregation? It appears that in the evolution of the two-chromosome genome of V. cholerae, either the secondary chromosome adapted to the main chromosome to ensure its maintenance or it is maintained independently, as are bacterial plasmids. An understanding of chromosome coordination is expected to bear on the evolutionary process of chromosome acquisition and on the efficacy of possible strategies for selective elimination of a pathogen by targeting a specific chromosome.

Enhanced recombinant protein productivity by genome reduction in Bacillus subtilis.

The emerging field of synthetic genomics is expected to facilitate the generation of microorganisms with the potential to achieve a sustainable society. One approach towards this goal is the reduction of microbial genomes by rationally designed deletions to create simplified cells with predictable behavior that act as a platform to build in various genetic systems for specific purposes. We report a novel Bacillus subtilis strain, MBG874, depleted of 874 kb (20%) of the genomic sequence. When compared with wild-type cells, the regulatory network of gene expression of the mutant strain is reorganized after entry into the transition state due to the synergistic effect of multiple deletions, and productivity of extracellular cellulase and protease from transformed plasmids harboring the corresponding genes is remarkably enhanced. To our knowledge, this is the first report demonstrating that genome reduction actually contributes to the creation of bacterial cells with a practical application in industry. Further systematic analysis of changes in the transcriptional regulatory network of MGB874 cells in relation to protein productivity should facilitate the generation of improved B. subtilis cells as hosts of industrial protein production.

Two separate DNA sequences within oriC participate in accurate chromosome segregation in Bacillus subtilis.

Current views of bacterial chromosome segregation vary in respect of the likely presence or absence of an active segregation mechanism involving a mitotic-like apparatus. Furthermore, little is known about cis-acting elements for chromosome segregation in bacteria. In this report, we show that two separate DNA regions, a 3' coding region of dnaA and the AT-rich sequence between dnaA and dnaN (the initial opening site of duplex DNA during replication), are necessary for efficient segregation of the chromosome in Bacillus subtilis. When a plasmid replicon was integrated into argG, far from oriC, on the chromosome and then the oriC function was disrupted, the oriC-deleted mutant formed anucleate cells at 5% possibly because of defects in chromosome segregation. However, when the two DNA sequences were added near oriN, frequency of anucleate cells decreased to 1%. In these cells, the origin (argG) regions were localized near cell poles, whereas they were randomly distributed in cells without the two DNA sequences. These results suggest that the two DNA sequences in and downstream of the dnaA gene participate in correct positioning of the replication origin region within the cell and that this function is associated with accurate chromosome segregation in B. subtilis.