Strain DR1 Research Articles

Amino Acid-Dependent Alterations in Cell Wall and Cell Morphology of Deinococcus indicus DR1.

Deinococcus radiodurans exhibits growth medium-dependent morphological variation in cell shape, but there is no evidence whether this phenomenon is observed in other members of the Deinococcaceae family. In this study, we isolated a red-pigmented, aerobic, Deinococcus indicus strain DR1 from Dadri wetland, India. This D. indicus strain exhibited cell–morphology transition from rod-shaped cells to multi-cell chains in a growth-medium-dependent fashion. In response to addition of 1% casamino acids in the minimal growth medium, rod-shaped cells formed multi-cell chains. Addition of all 20 amino acids to the minimal medium was able to recapitulate the phenotype. Specifically, a combination of L-methionine, L-lysine, L-aspartate, and L-threonine caused morphological alterations. The transition from rod shape to multi-cell chains is due to delay in daughter cell separation after cell division. Minimal medium supplemented with L-ornithine alone was able to cause cell morphology changes. Furthermore, a comparative UPLC analysis of PG fragments isolated from D. indicus cells propagated in different growth media revealed alterations in the PG composition. An increase in the overall cross-linkage of PG was observed in muropeptides from nutrient-rich TSB and NB media versus PYE medium. Overall our study highlights that environmental conditions influence PG composition and cell morphology in D. indicus.

Draft Genome Sequence of Deinococcus indicus DR1, a Novel Strain Isolated from a Freshwater Wetland.

ABSTRACTDeinococcus indicus strain DR1, a red-pigmented, arsenic- and radiation-resistant bacterium, was isolated from a water sample of the Dadri wetland, Uttar Pradesh, India. Here, we report a draft genome sequence of this strain, which may provide useful information regarding the genes and pathways involved in heavy-metal bioremediation.

Effects of phosphoenolpyruvate carboxylase desensitization on glutamic acid production in Corynebacterium glutamicum ATCC 13032

Phosphoenolpyruvate carboxylase (PEPC) in Corynebacterium glutamicum ATCC13032, a glutamic-acid producing actinobacterium, is subject to feedback inhibition by metabolic intermediates such as aspartic acid and 2-oxoglutaric acid, which implies the importance of PEPC in replenishing oxaloacetic acid into the TCA cycle. Here, we investigated the effects of feedback-insensitive PEPC on glutamic acid production. A single amino-acid substitution in PEPC, D299N, was found to relieve the feedback control by aspartic acid, but not by 2-oxoglutaric acid. A simple mutant, strain R1, having the D299N substitution in PEPC was constructed from ATCC 13032 using the double-crossover chromosome replacement technique. Strain R1 produced glutamic acid at a concentration of 31.0g/L from 100g/L glucose in a jar fermentor culture under biotin-limited conditions, which was significantly higher than that of the parent, 26.0g/L (1.19-fold), indicative of the positive effect of desensitized PEPC on glutamic acid production. Another mutant, strain DR1, having both desensitized PEPC and PYK-gene deleted mutations, was constructed in a similar manner using strain D1 with a PYK-gene deleted mutation as the parent. This mutation had been shown to enhance glutamic acid production in our previous study. Although marginal, strain D1 produced higher glutamic acid, 28.8g/L, than ATCC13032 (1.11-fold). In contrast, glutamic acid production by strain DR-1 was elevated up to 36.9g/L, which was 1.42-fold higher than ATCC13032 and significantly higher than the other three strains. The results showed a synergistic effect of these two mutations on glutamic acid production in C.glutamicum.

Acinetobacter species as model microorganisms in environmental microbiology: current state and perspectives.

Acinetobacter occupies an important position in nature because of its ubiquitous presence in diverse environments such as soils, fresh water, oceans, sediments, and contaminated sites. Versatile metabolic characteristics allow species of this genus to catabolize a wide range of natural compounds, implying active participation in the nutrient cycle in the ecosystem. On the other hand, multi-drug-resistant Acinetobacter baumannii causing nosocomial infections with high mortality has been raising serious concerns in medicine. Due to the ecological and clinical importance of the genus, Acinetobacter was proposed as a model microorganism for environmental microbiological studies, pathogenicity tests, and industrial production of chemicals. For these reasons, Acinetobacter has attracted significant attention in scientific and biotechnological fields, but only limited research areas such as natural transformation and aromatic compound degradation have been intensively investigated, while important physiological characteristics including quorum sensing, motility, and stress response have been neglected. The aim of this review is to summarize the recent achievements in Acinetobacter research with a special focus on strain DR1 and to compare the similarities and differences between species or other genera. Research areas that require more attention in future research are also suggested.

Complexity of cell–cell interactions between Pseudomonas sp. AS1 and Acinetobacter oleivorans DR1: metabolic commensalism, biofilm formation and quorum quenching

Acinetobacter oleivorans DR1 lacks an upper pathway for naphthalene degradation and cannot grow using naphthalene as sole carbon source; however, it is capable of growing under naphthalene-amended conditions in the presence of naphthalene-degrading Pseudomonas sp. AS1. 1H-NMR spectroscopy, high-performance liquid chromatography and gene expression analyses showed that salicylate is a major secreted metabolic intermediate during naphthalene degradation by strain AS1 and that, in turn, it supports the growth of strain DR1. Interspecies biofilm formation, monitored using confocal laser scanning microscopy and microtiter assays, demonstrated that biofilm formation by strain AS1 increased dramatically in the presence of strain DR1 because of the exopolysaccharides generated by the latter. Furthermore, the metabolic commensal interaction of the two strains altered the initial attachment behavior of strain DR1 during biofilm formation. When this strain was cultivated alone under naphthalene-amended conditions, the cells immediately attached to the surface, probably due to the absence of usable substrates, whereas similar behavior was not observed in the mixed culture. This interspecies cell–cell interaction became more complex due to quenching of the quorum-sensing signal of strain DR1 by strain AS1. These complex metabolic and physiological interactions observed in mixed cultures suggest that interspecies interaction is more complicated than previously surmised.

Comparative Genomic Analysis of Acinetobacter oleivorans DR1 To Determine Strain-Specific Genomic Regions and Gentisate Biodegradation

The comparative genomics of Acinetobacter oleivorans DR1 assayed with A. baylyi ADP1, A. calcoaceticus PHEA-2, and A. baumannii ATCC 17978 revealed that the incorporation of phage-related genomic regions and the absence of transposable elements have contributed to the large size (4.15 Mb) of the DR1 genome. A horizontally transferred genomic region and a higher proportion of transcriptional regulator- and signal peptide-coding genes were identified as characteristics of the DR1 genome. Incomplete glucose metabolism, metabolic pathways of aromatic compounds, biofilm formation, antibiotics and metal resistance, and natural competence genes were conserved in four compared genomes. Interestingly, only strain DR1 possesses gentisate 1,2-dioxygenase (nagI) and grows on gentisate, whereas other species cannot. Expression of the nagI gene was upregulated during gentisate utilization, and four downstream open reading frames (ORFs) were cotranscribed, supporting the notion that gentisate metabolism is a unique characteristic of strain DR1. The genomic analysis of strain DR1 provides additional insights into the function, ecology, and evolution of Acinetobacter species.

Physiological and metabolic responses for hexadecane degradation in Acinetobacter oleivorans DR1

The hexadecane degradation of Acinetobacter oleivorans DR1 was evaluated with changes in temperature and ionic salt contents. Hexadecane degradation of strain DR1 was reduced markedly by the presence of sodium chloride (but not potassium chloride). High temperature (37°C) was also shown to inhibit the motility, biofilm formation, and hexadecane biodegradation. The biofilm formation of strain DR1 on the oil-water interface might prove to be a critical physiological feature for the degradation of hexadecane. The positive relationship between biofilm formation and hexadecane degradation could be observed at 30° C, but not at low temperatures (25°C). Alterations in cell hydrophobicity and EPS production by temperature and salts were not correlated with biofilm formation and hexadecane degradation. Our proteomic analyses have demonstrated that metabolic changes through the glyoxylate pathway are important for efficient degradation of hexadecane. Proteins involved in fatty acid metabolism, gluconeogenesis, and oxidative stress defense proteins appear to be highly expressed during biodegradation of hexadecane. These results suggested that biofilm formation and oxidative stress defense are important physiological responses for hexadecane degradation along with metabolic switch to glyoxylate pathway in strain DR1.

Acinetobacter oleivorans sp. nov. Is capable of adhering to and growing on diesel-oil

A diesel-oil and n-hexadecane-degrading novel bacterial strain, designated DR1(T), was isolated from a rice paddy in Deok-So, South Korea. The strain DR1(T) cells were Gram-negative, aerobic coccobacilli, and grew at 20-37°C with the optimal temperature of 30°C, and an optimal pH of 6-8. Interestingly, strain DR1(T) was highly motile (swimming and swarming motility) using its fimbriae, and generated N-acyl homoserine lactones as quorum-sensing signals. The predominant respiratory quinone as identified as ubiquinone-9 (Q-9) and DNA G+C content was 41.4 mol%. Comparative 16S rRNA gene sequence-based phylogenetic analysis placed the strain in a clade with the species A. calcoaceticus, A. haemolyticus, A. baumannii, A. baylyi, and A. beijerinckii, with which it evidenced sequence similarities of 98.2%, 97.4%, 97.2%, 97.1%, and 97.0%, respectively. DNA-DNA hybridization values between strain DR1(T) and other Acinetobacter spp. were all less than 20%. The physiological and taxonomic characteristics with the DNA-DNA hybridization data supported the identification of strain DR1(T) in the genus Acinetobacter as a novel species, for which the name Acinetobacter oleivorans sp. nov. is proposed. The type strain is DR1(T) (=KCTC 23045(T) =JCM 16667(T)).

Complete Genome Sequence of the Diesel-Degrading Acinetobacter sp. Strain DR1

The genus Acinetobacter is ubiquitous in soil, aquatic, and sediment environments and includes pathogenic strains, such as A. baumannii. Many Acinetobacter species isolated from various environments have biotechnological potential since they are capable of degrading a variety of pollutants. Acinetobacter sp. strain DR1 has been identified as a diesel degrader. Here we report the complete genome sequence of Acinetobacter sp. DR1 isolated from the soil of a rice paddy.

Contribution of quorum-sensing system to hexadecane degradation and biofilm formation in Acinetobacter sp. strain DR1

To investigate roles of quorum-sensing (QS) system in Acinetobacter sp. strain DR1 and rifampicin-resistant variant (hereinafter DR1R). The DR1 strain generated three putative acyl homoserine lactones (AHLs), while the DR1R produced only one signal and QS signal production was abrogated in the aqsI (LuxI homolog) mutant. The hexadecane-degradation and biofilm-formation capabilities of DR1, DR1R, and aqsI mutants were compared, along with their proteomic data. Proteomics analysis revealed that the AHL lactonase responsible for degrading QS signal was highly upregulated in both DR1R and aqsI mutant, also showed that several proteins, including ppGpp synthase, histidine kinase sensors, might be under the control of QS signalling. Interestingly, biofilm-formation and hexadecane-biodegradation abilities were reduced more profoundly in the aqsI mutant. These altered phenotypes of the aqsI mutant were restored via the addition of free wild-type cell supernatant and exogenous C(12) -AHL. The QS system in strain DR1 contributes to hexadecane degradation and biofilm formation. This is the first report to demonstrate that a specific QS signal appears to be a critical factor for hexadecane degradation and biofilm formation in Acinetobacter sp. strain DR1.

Phenotypic and Physiological Changes in Acinetobacter sp. Strain DR1 with Exogenous Plasmid

The genus Acinetobacter has been recognized to take up exogenous DNA from the environment. In this study, we conducted natural transformation with a novel diesel-degrading Acinetobacter sp. strain, designated strain DR1, using the broad host range plasmid pRK415. Many factors, including temperature, quantities of DNA, and aeration have proven critically important for efficient natural transformation. Interestingly, the Acinetobacter sp. strain DR1 (pRK415) differed both phenotypically and physiologically from the wild-type strain in several regards, including motility, biofilm formation ability, and responses to oxidative stress: the transformed cells were rendered more sensitive to hydrogen peroxide and cumene hydroperoxide, and their motilities and biofilm formation activity were also attenuated. Our data demonstrated that caution should be exercised when conducting genetic manipulation with plasmids, due to the possibility that phenotypic and physiological changes in the host might occur along with the uptake of plasmids.

Trade‐off between antibiotic resistance and biological fitness in Acinetobacter sp. strain DR1

Rifampicin, a bactericidal antibiotic drug, is routinely used to make an environmental recipient selective in laboratory-conjugation experiments. We noticed, inadvertently, that the rifampicin-resistant Acinetobacter sp. strain DR1, a recently discovered hexadecane-degrading environmental isolate, exhibited a substantial loss of quorum sensing signalling. The domesticated ampicillin-resistant strain, DR1, evidenced more dramatic phenotypic changes than were observed in the rifampicin-resistant cells: a complete loss of quorum sensing, a loss in swimming and swarming motilities, poor fimbrial expression, increased rigidity in membrane fatty acid composition and reduced hexadecane degradation capability. Interestingly, the motility of strain DR1 grown adjacent to a streptomycin-producing Streptomyces griceus was permanently abrogated, where this change was heritable and other phenotypic changes could not be detected. In this study, we have reported for the first time that the in situ acquisition of antibiotic resistance may reduce biological fitness, including losses in the production of quorum sensing signals, motility and substrate utilization, and each antibiotic is associated with different degrees of phenotypic and genetic alterations. Our data also suggested that the domestication of environmental isolates should be approached with caution, as there are phenotypic variations in antibiotic-resistant cells that might not be noticeable unless all possible phenotypic assays are conducted.

Protection against diesel oil toxicity by sodium chloride-induced exopolysaccharides in Acinetobacter sp. strain DR1

Acinetobacter sp. strain DR1 is capable of growth on diesel oil. Interestingly, the degradation of diesel oil by the strain DR1 is enhanced in the presence of sodium chloride (NaCl). However, the growth rate of strain DR1 is not affected by the presence of NaCl. Northern blot analysis has also demonstrated that the effect of NaCl on the degradation of diesel oil is not attributable to increased levels of alkane hydroxylase (AlkM-type) gene expression. Rather, we have noted an increase in the exopolysaccharide (EPS) yields of strain DR1 under high NaCl conditions (9-fold). The lag-time of diesel oil biodegradation was significantly shorter in the strain DR1 with exogenous EPS than in the controls, although EPS alone does not support the growth of strain DR1. The recovery of strain DR1 when exposed to diesel oil was accelerated when exogenous EPS was added to the medium. The overproduction of EPS was also noted in the presence of diesel oil and n-hexadecane. The data indicated that EPS overproduction might play a protective role against diesel oil toxicity. Along with the results of the soil microcosm tests, the data presented herein demonstrated that NaCl-induced EPS is associated with a reduction in diesel oil toxicity, and thus increases diesel oil biodegradation in Acinetobacter sp. strain DR1.

Geographical Distribution and Diversity of Moderately Thermophilic Members of the Thermoplasmatales

Oxidation of ferrous iron by moderately thermophilic species of the genus Ferroplasma is of considerable potential value in commercial bioleaching operations. A collection of strains was enriched and isolated from a number of natural or industrial acidic sites at 45 and 55°C. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that all strains were members of the Order Thermoplasmatales. Stains BH8 and BH12 showed 98% sequence similarity with Ferroplasma acidiphilum strain YT and DR1 respectively. Strains BH7 and BH10 were closely related to the moderately thermophilic species Ferroplasma cupricumulans. Strain BH9, a moderately thermophilic organotroph, was unrelated to previously described species and probably represents a novel genus. Strains BH8 and BH12 showed some unique physiological differences to the type species F. acidiphilum. Unlike F. acidiphilum, both strains were moderately thermophilic with a temperature range for strains BH8 and BH12 of 24 to 61°C and 27 to 49°C respectively. Strain BH12 grew organotrophically on Darland’s Medium containing glucose and yeast extract and chemomixotrophically on Darland’s Medium supplemented with ferrous sulphate. Moderately thermophilic species of Ferroplasma and Ferroplasma-like genera appear widely distributed geographically and possess considerable physiological and phylogenetic diversity that may benefit industrial bioleaching processes.

The putative transcriptional repressor McbR, member of the TetR-family, is involved in the regulation of the metabolic network directing the synthesis of sulfur containing amino acids in Corynebacterium glutamicum

In order to isolate transcriptional regulatory proteins involved in l-methionine-dependent repression in Corynebacterium glutamicum, proteins binding to the putative promoter region upstream of the metY gene were isolated by DNA affinity chromatography. One of the isolated proteins was identified as a putative transcriptional repressor of the TetR-family by a mass spectrometry fingerprint technique based on the complete C. glutamicum genome sequence. The respective gene, designated mcbR, was deleted in the mutant strain C. glutamicum DR1. Using 2D-PAGE, the protein contents of the C. glutamicum wild type and the mutant strain DR1 grown in media with or without l-methionine supplementation were compared and a set of six proteins was identified. Their abundance was drastically enhanced in the mutant strain and no longer influenced by l-methionine added to the growth medium. The corresponding genes were identified by mass spectrometry fingerprint analysis. They included metY encoding O-acetyl- l-homoserine sulfhydrylase, metK encoding S-adenosyl-methionine synthethase, hom encoding homoserine dehydrogenase, cysK encoding l-cysteine synthase, cysI encoding an NADPH dependant sulfite reductase, and ssuD encoding an alkanesulfonate monooxygenase. Evidently, the putative transcriptional repressor McbR is involved in the regulation of the metabolic network directing the synthesis of l-methionine in C. glutamicum. The C. glutamicum mcbR mutant can be considered to represent a first step in the construction of an l-methionine production strain.