Phenotypic Adaptations Help Rhodococcus erythropolis Cells during the Degradation of Paraffin Wax.

During crude oil extraction, the reduction in temperature and pressure results in the precipitation of paraffin wax that contains 20-40 carbon chain hydrocarbons. The paraffin wax may accumulate inside production tubes, pipelines, and processing facilities, and also in tankers during petroleum transportation. There are few bacterial strains that are able to degrade solid substrates. In the present study, the biodegradation of paraffin is evaluated using Rhodococcus erythropolis cells. This bacterium is able to grow using paraffin wax from an oil refinery plant as the sole carbon source. The cells grow as a thick biofilm over the solid substrate, make scale-like structures that increase the area of the initially smooth surface of paraffin, produce biosurfactants, and become more negatively charged than ethanol- or glucose-grown cells. When paraffin wax is supplied as microparticles, to increase the cell-substrate contact area and to simulate paraffin precipitation, the cells also adjust the composition of the fatty acids of the phospholipids of the cellular membrane to decrease its fluidity and paraffin biodegradation increases considerably. The study suggests that the phenotypic adaptation of R. erythropolis cells may be used to degrade paraffin wax under real conditions.

Biocontrol of Soft Rot: Confocal Microscopy Highlights Virulent Pectobacterial Communication and Its Jamming by Rhodococcal Quorum-Quenching.

Confocal laser-scanning microscopy was chosen to observe the colonization and damage caused by the soft rot Pectobacterium atrosepticum and the protection mediated by the biocontrol agent Rhodococcus erythropolis. We developed dual-color reporter strains suited for monitoring quorum-sensing and quorum-quenching activities leading to maceration or biocontrol, respectively. A constitutively expressed cyan or red fluorescent protein served as a cell tag for plant colonization, while an inducible expression reporter system based on the green fluorescent protein gene enabled the simultaneous recording of signaling molecule production, detection, or degradation. The dual-colored pathogen and biocontrol strains were used to coinoculate potato tubers. At cellular quorum, images revealed a strong pectobacterial quorum-sensing activity, especially at the plant cell walls, as well as a concomitant rhodococcal quorum-quenching response, at both the single-cell and microcolony levels. The generated biosensors appear to be promising and complementary tools useful for molecular and cellular studies of bacterial communication and interference.

Modeling Escherichia coli and Rhodococcus erythropolis transport through wettable and water repellent porous media.

Water protection and bioremediation strategies in the vadose zone require understanding the factors controlling bacterial transport for different hydraulic conditions. Breakthrough experiments were made in two different flow conditions: i) an initial bacteria pulse under ponded infiltration into dry sand (-15,000 cm); ii) a second bacteria pulse into the same columns during subsequent infiltration in constant water content and steady-state flow. Escherichia coli (E. coli) and Rhodococcus erythropolis (R. erythropolis) were used to represent hydrophilic and hydrophobic bacteria, respectively. Equilibrium and attachment/detachment models were tested to fit bromide (Br-) and bacteria transport data using HYDRUS-1D. Derjaguin-Landau-Verwey-Overbeek (DLVO) and extended DVLO (XDLVO) interaction energy profiles were calculated to predict bacteria sorption at particles. Adsorption of bacteria at air-water interfaces was estimated by a hydrophobic force approach. Results suggested greater retention of bacteria in water repellent sand compared with wettable sand. Inverse parameter optimization suggested that physico-chemical attachment of both E. coli and R. erythropolis was thousands of times lower in wettable than repellant sand and straining was 10-fold lower in E. coli for wettable vs repellant sand compared to the exact opposite by orders of magnitude with R. erythropolis. HYDRUS did not provide a clear priority of importance of solid-water or air-water interfaces in bacteria retention. Optimized model parameters did not show a clear relation to the (X)DLVO adsorption energies. This illustrated the ambivalence of (X)DLVO to predict bacterial attachment at solid soil particles of different wetting properties. Simultaneous analysis of mass recovery, numerical modeling, and interaction energy profiles thus suggested irreversible straining due to bacteria sizing as dominant compared to attachment to liquid-solid or liquid-air interfaces. Further studies are needed to distinguish straining mechanisms (i.e. pore structure or film straining) in different hydraulic conditions.

Detection and Discrimination of Bacterial Colonies with Mueller Matrix Imaging.

The polarization imaging technique is a powerful approach to probe microstructural and optical information of biological structures (e.g., tissue samples). Here, we have studied the polarization properties of different bacterial colonies in order to evaluate the possibility of bacterial detection and discrimination. In this regard, we have taken the backscattering Mueller matrix images of four different bacteria colonies (i.e., Escherichia coli, Lactobacillus rhamnosus, Rhodococcus erythropolis, and Staphylococcus aureus). Although the images have the potential to distinguish qualitatively different bacterial colonies, we explored more accurate and quantitative parameters criteria for discrimination of bacterial samples; more specifically, we have exploited the Mueller matrix polar decomposition (MMPD),frequency distribution histogram (FDH), and central moment analysis method. The outcomes demonstrated a superior capacity of Mueller matrix imaging, MMPD, and FDH in bacterial colonies identification and discrimination. This approach might pave the way for a reliable, efficient, and cheap way of identification of infectious diseases.

Biodegradation and chemotaxis of polychlorinated biphenyls, biphenyls, and their metabolites by Rhodococcus spp.

Two biphenyl-degrading bacterial strains, SS1 and SS2, were isolated from polychlorinated biphenyl (PCB)-contaminated soil. They were identified as Rhodococcus ruber and Rhodococcus pyridinivorans based on the 16S rRNA gene sequence, as well as morphological, physiological and biochemical characteristics. SS1 and SS2 exhibited tolerance to 2000 and 3000 mg/L of biphenyl. And they could degrade 83.2 and 71.5% of 1300 mg/L biphenyl within 84 h, respectively. In the case of low-chlorinated PCB congeners, benzoate and 3-chlorobenzoate, the degradation activities of SS1 and SS2 were also significant. In addition, these two strains exhibited chemotactic response toward TCA-cycle intermediates, benzoate, biphenyl and 2-chlorobenzoate. This study indicated that, like the flagellated bacteria, non-flagellated Rhodococcus spp. might actively seek substrates through the process of chemotaxis once the substrates are depleted in their surroundings. Together, these data provide supporting evidence that SS1 and SS2 might be good candidates for restoring biphenyl/PCB-polluted environments.

Highly chemoselective and efficient production of 2,6-difluorobenzamide using Rhodococcus ruber CGMCC3090 resting cells.

Chemoselective biocatalytic hydrolysis of nitriles is a valuable alternative to chemical hydrolysis that operates under harsh conditions. 2,6-Difluorobenzamide (DFBAM) is an essential intermediate derived from synthesis of benzoyl urea insecticide from 2,6-difluorobenzonitrile (DFBN). High yield of DFBAM was achieved, and the method using resting cells of Rhodococcus ruber CGMCC3090 exhibited excellent product specificity. The reaction parameters for DFBAM biosynthesis were also investigated. The resting cells effectively converted DFBN at high concentrations of up to 3.5 mol L-1 without forming acids or other by-products. Therefore, biological production of DFBAM features high yield, chemoselectivity, low cost, and environment friendliness and is more suitable to the industry than chemical synthesis techniques.

Tuning and elucidation of the colony dimorphism in Rhodococcus ruber associated with cell flocculation in large scale fermentation.

Prevention of cell flocculation in large-scale fermentation is of great importance for most industrial microbes. Using Rhodococcus ruber TH3 as a model strain, we revealed that the undesired cell flocculation in a fermenter was associated with the colony dimorphism phenomenon, and it only occurred in the rough-type of cells (R-TH3) instead of the smooth-type of cells (S-TH3). By analyzing the transcriptome differences of R-TH3 and S-TH3, six representative genes with significantly upregulated transcription in S-TH3 were selected and overexpressed in R-TH3. The colony morphotypes of the six engineered strains changed to different extents, in which overexpressions of three lipid metabolism-related proteins LM1, LM2, and LM3 tuned the colony morphotype from rough to almost as smooth as in S-TH3. SEM observation confirmed the cell surface difference of the engineered strains from R-TH3. Their cell surface hydrophobicity also reduced, and the cell sedimentation behaviors were consequently changed as expected. Using R-TH3/LM1 as the representative of the engineered bacteria, fatty acids of the cell envelopes were measured. Fatty acid contents of S-TH3, R-TH3/LM1, and R-TH3 were 27.21, 24.10, and 22.24%, respectively. Among all the fatty acids, stearic acid binding to hydrophilic extracellular polysaccharides (EPS) in Rhodococcus showed significant differences among the cells. The EPS contents of S-TH3, R-TH3/LM1, and R-TH3 were 191, 163, and 137 mg/g cells. Hence, the hydrophilicity of the S-TH3 cells was mainly due to the EPS in the outermost layer of the cells. Increase of fatty acids especially stearic acid results in the increase of the bound EPS, finally bringing about the hydrophilicity enhancement.

Localization of Low Copy Number Plasmid pRC4 in Replicating Rod and Non-Replicating Cocci Cells of Rhodococcus erythropolis PR4.

Rhodococcus are gram-positive bacteria, which can exist in two different shapes rod and cocci. A number of studies have been done in the past on replication and stability of small plasmids in this bacterium; however, there are no reports on spatial localization and segregation of these plasmids. In the present study, a low copy number plasmid pDS3 containing pRC4 replicon was visualized in growing cells of Rhodococcus erythropolis PR4 (NBRC100887) using P1 parS-ParB-GFP system. Cells were initially cocci and then became rod shaped in exponential phase. Cocci cells were found to be non-replicating as evident by the presence of single fluorescence focus corresponding to the plasmid and diffuse fluorescence of DnaB-GFP. Rod shaped cells contained plasmid either present as one fluorescent focus observed at the cell center or two foci localized at quarter positions. The results suggest that the plasmid is replicated at the cell center and then it goes to quarter position. In order to observe the localization of plasmid with respect to nucleoid, plasmid segregation was also studied in filaments where it was found to be replicated at the cell center in a nucleoid free region. To the best of our knowledge, this is the first report on segregation of small plasmids in R. erythropolis.

Induction of Viable but Nonculturable State in Rhodococcus and Transcriptome Analysis Using RNA-seq.

Viable but nonculturable (VBNC) bacteria, which maintain the viability with loss of culturability, universally exist in contaminated and non-contaminated environments. In this study, two strains, Rhodococcus sp. TG13 and TN3, which were isolated from PCB-contaminated sediment and non-contaminated sediment respectively, were investigated under low temperature and oligotrophic conditions. The results indicated that the two strains TG13 and TN3 could enter into the VBNC state with different incubation times, and could recover culturability by reversal of unfavourable factors and addition of resuscitation-promoting factor (Rpf), respectively. Furthermore, the gene expression variations in the VBNC response were clarified by Illumina high throughput RNA-sequencing. Genome-wide transcriptional analysis demonstrated that up-regulated genes in the VBNC cells of the strain TG13 related to protein modification, ATP accumulation and RNA polymerase, while all differentially expressed genes (DEGs) in the VBNC cells of the strain TN3 were down-regulated. However, the down-regulated genes in both the two strains mainly encoded NADH dehydrogenase subunit, catalase, oxidoreductase, which further verified that cold-induced loss of ability to defend oxidative stress may play an important role in induction of the VBNC state. This study further verified that the molecular mechanisms underlying the VBNC state varied with various bacterial species. Study on the VBNC state of non-pathogenic bacteria will provide new insights into the limitation of environmental micro-bioremediation and the cultivation of unculturable species.

Lipid accumulation by Rhodococcus rhodochrous grown on glucose.

Biodiesel is an alternative fuel made from costly vegetable oil feedstocks. Some microorganisms can accumulate lipids when nutrients are limited and carbon is in excess. Rhodococcus rhodochrous is a gram-positive bacterium most often used in bioremediation or acrylamide production. The purpose of this study was to investigate and characterize the lipid accumulation capabilities of R. rhodochrous. Shake flasks and a large-scale fermentation were used to cultivate R. rhodochrous in varying concentrations of glucose. R. rhodochrous achieved almost 50 % of dry cell mass as lipid when grown in 20 g/L of glucose. Wax esters and triglycerides were identified in R. rhodochrous lipid extract. The transesterified extractables of R. rhodochrous consisted of mostly palmitic (35 %) and oleic (42 %) acid methyl esters. This study shows R. rhodochrous to be an oleaginous bacterium with potential for application in alternative fuels.