[Analysis of Maximum Growth Rate and Construction of Predictive Growth Model for Bacillus cereusin Mashed Potato by Calorimetric Method].

The maximum growth rate (µmax) of Bacillus cereus was estimated using a non-destructive isothermal calorimetric method, and a growth prediction model was constructed based on the measurement results. SCD medium and mashed potato were inoculated with serial-diluted inoculum of B. cereus. Heat generation curves were determined using an isothermal calorimeter at 35, 25, and 15℃. The µmax was determined from the relationship between the increase in B. cereus cell number and incubation time, which was detected through the heat generation of the B. cereus biological process. Moreover, the growth prediction model was constructed using Ratkowsky's square-root model. The results of the growth prediction model based on the data of the calorimetric and conventional culture methods for SCD were expressed as √µCalmax=0.0354 (T-4.9)[R2=0.99] and √µCCMmax=0.0335 (T-5.0)[R2=0.99]; a similar equation was provided by both methods. Conversely, the results of the growth prediction model based on the calorimetric method data for mashed potato were given as √µCalmax=0.0390 (T-8.5)[R2=0.99]; the maximum growth rates at 30 and 20℃ were predicted as 0.70 and 0.20 (1/hr), respectively. The maximum growth rates obtained using the conventional culture method were 0.63 and 0.29 (1/hr), respectively, similar to the calorimetric method results. The predictive microbiological analysis using the calorimetric method enabled the rapid provision of a growth prediction equation, and the number of samples could be substantially reduced compared with that for the conventional culture method.

Draft Genome Sequence of Pseudomonas sp. Strain LLC-1 (NBRC 111237), Capable of Metabolizing Lignin-Derived Low-Molecular-Weight Compounds.

Pseudomonas sp. strain LLC-1 (NBRC 111237), isolated from soil, metabolizes lignin-derived low-molecular-weight compounds and utilizes vanillin and vanillic acid as its sole sources of carbon. Here, we report the draft genome sequence of Pseudomonas sp. strain LLC-1.

Amino acid residues critical for DNA binding and inducer recognition in CbnR, a LysR-type transcriptional regulator from Cupriavidus necator NH9.

CbnR, a LysR-type transcriptional regulator from Cupriavidus necator NH9, activates the transcription of chlorocatechol-degradative enzymes. To activate the transcription, CbnR needs to bind not only to the cbnA promoter but also to the inducer. In this study, the transcriptional activity and DNA-binding activity of twenty-five mutants of CbnR were analyzed. Of the 17 mutants of the DNA-binding domain, 11 mutants lost their ability to activate transcription. While most mutants without transcriptional activation did not show DNA-binding activity, Asn17Ala, Gln29Ala, and Pro30Ala retained DNA-binding activity, suggesting that transcriptional activation by CbnR requires more than its binding to promoter DNA. Of the 8 mutants of the regulatory domain, 6 mutants changed their responses to the inducer, when compared with wild-type CbnR. Interestingly, Arg199Ala and Val246Ala induced constitutive expression of the cbnA promoter without the inducer, suggesting that these mutations brought about a conformational change mimicking that induced by the inducer molecule.

Insights into the genomic plasticity of Pseudomonas putida KF715, a strain with unique biphenyl-utilizing activity and genome instability properties.

Pseudomonas putida KF715 exhibits unique properties in both catabolic activity and genome plasticity. Our previous studies revealed that the DNA region containing biphenyl and salycilate metabolism gene clusters (termed the bph-sal element) was frequently deleted and transferred by conjugation to closely related P. putida strains. In this study, we first determined the complete nucleotide sequence of the KF715 genome. Next, to determine the underlying cause of genome plasticity in KF715, we compared the KF715 genome with the genomes of one KF715 defective mutant, two transconjugants, and several P. putida strains available from public databases. The gapless KF715 genome sequence revealed five replicons: one circular chromosome, and four plasmids. Southern blot analysis indicated that most of the KF715 cell population carries the bph-sal element on the chromosome whereas a small number carry it on a huge plasmid, pKF715A. Moreover, the bph-sal element is present stably on the plasmid and did not integrate into the chromosome of its transconjugants. Comparative genome analysis and experiments showed that a number of diverse putative genetic elements are present in KF715 and are likely involved in genome rearrangement. These data provide insights into the genetic plasticity and adaptability of microorganisms for survival in various ecological niches.

Phylogenetic analysis reveals the taxonomically diverse distribution of the Pseudomonas putida group.

Pseudomonas putida is well-known for degradation activities for a variety of compounds and its infections have been reported. Thus, P. putida includes both clinical and nonclinical isolates. To date, no reports have examined the phylogenetic relationship between clinical and nonclinical isolates of the P. putida group. In this study, fifty-nine strains of P. putida group containing twenty-six clinical, and thirty-three nonclinical, isolates, were subjected to phylogenetic and taxonomic analyses based on 16S rRNA gene sequences and nine housekeeping gene sequences, including argS, dnaN, dnaQ, era, gltA, gyrB, ppnK, rpoB, and rpoD, to obtain insights into the diversity of species in this group. More than 97.6% similarity was observed among the 16S rRNA gene sequences of all the strains examined, indicating that the resolution of 16S rRNA gene sequences is inadequate. Phylogenetic analysis based on the individual housekeeping genes listed above improved the resolution of the phylogenetic trees, which are different from each other. Multilocus sequence analysis (MLSA) based on the concatenated sequences of the nine genes significantly improved the resolution of the phylogenetic tree, and yielded approximately the same results as average nucleotide identity (ANI) analysis, suggesting its high reliability. ANI analysis classified the fifty-nine strains into twenty-six species containing seventeen singletons and nine strain clusters based on the 95% threshold. It also indicated the mixed distribution of clinical and nonclinical isolates in the six clusters, suggesting that the genomic difference between clinical and nonclinical isolates of the P. putida group is subtle. The P. putida type strain NBRC 14164T is a singleton that is independently located from the P. putida strains distributed among the six clusters, suggesting that the classification of these strains and the differentiation of species in the P. putida group should be re-examined. This study greatly expands insights into the phylogenetic diversity of the P. putida group.

Draft Genome Sequence of Comamonas thiooxydans Strain PHE2-6 (NBRC 110656), a Chlorinated-Ethene-Degrading Bacterium.

Comamonas thiooxydans strain PHE2-6 (NBRC 110656), which was isolated from a trichloroethene-contaminated site in Japan, utilizes phenol as a sole source of carbon and cometabolizes cis- and trans-dichloroethenes. We report here the draft genome sequence of this strain, containing 5,309,680 bp, with 60.6% G+C content.

Isolation and characterization of a bacterial strain that degrades cis-dichloroethenein the absence of aromatic inducers.

Bacteria capable of degrading cis-dichloroethene (cDCE) were screened from cDCE-contaminated soil, and YKD221, a bacterial strain that exhibited a higher growth on minimal salt agar plates in the presence of cDCE than in the absence of cDCE, were isolated. Phylogenetic studies of the 16S rRNA as well as gyrB, rpoD, and recA in YKD221 indicated that this strain is closely related to the type strains of Pseudomonas plecoglossicida, monteilii, and putida. An average nucleotide identity analysis indicated that YKD221 is most closely related to P. putida strains, including the type strain, which suggests that YKD221 belongs to P. putida. Although the genome of YKD221 was very similar to that of P. putida F1, a toluene-degrading strain, the YKD221 genome has 15 single-nucleotide polymorphisms and 4 insertions compared with the F1 genome. YKD221 caused the release of sufficient chloride ions from cDCE to suggest that the strain is able to completely dechlorinate and degrade cDCE. YKD221 also degraded trichloroethene but was unable to degrade trans-dichloroethene and tetrachloroethene. The degradation activity of YKD221 was elevated after growth on toluene. Inactivation of todC1, which encodes for a large subunit of the catalytic terminal component in toluene dioxygenase, resulted in a complete loss of growth on toluene and cDCE degradation activity. This is the first evidence of the involvement of todC1C2BA-coded toluene dioxygenase in cDCE degradation. YKD221 did not appear to grow on cDCE in a minimal salt liquid medium. However, YKD221 did exhibit an enhanced increase in cell concentration and volume of cells during growth on minimal salt agar plates with cDCE when first grown in LB medium. This behavior appears to have led us to misinterpret our initial results on YKD221 as an indication of improved growth in the presence of cDCE.

Draft Genome Sequence of a Chlorinated-Ethene Degrader, Cupriavidus necator Strain PHE3-6 (NBRC 110655).

Cupriavidus necator strain PHE3-6 grows on phenol as a sole carbon source and cometabolizes cis- and trans-dichloroethenes and trichloroethene. Here, we report the draft genome sequence of PHE3-6, which provides insights into the degradation system of phenol and chlorinated ethenes.

Identification of novel extracellular protein for PCB/biphenyl metabolism in Rhodococcus jostii RHA1.

Rhodococcus jostii RHA1 (RHA1) degrades polychlorinated biphenyl (PCB) via co-metabolism with biphenyl. To identify the novel open reading frames (ORFs) that contribute to PCB/biphenyl metabolism in RHA1, we compared chromatin immunoprecipitation chip and transcriptomic data. Six novel ORFs involved in PCB/biphenyl metabolism were identified. Gene deletion mutants of these 6 ORFs were made and were tested for their ability to grow on biphenyl. Interestingly, only the ro10225 deletion mutant showed deficient growth on biphenyl. Analysis of Ro10225 protein function showed that growth of the ro10225 deletion mutant on biphenyl was recovered when exogenous recombinant Ro10225 protein was added to the culture medium. Although Ro10225 protein has no putative secretion signal sequence, partially degraded Ro10225 protein was detected in conditioned medium from wild-type RHA1 grown on biphenyl. This Ro10225 fragment appeared to form a complex with another PCB/biphenyl oxidation enzyme. These results indicated that Ro10225 protein is essential for the formation of the PCB/biphenyl dioxygenase complex in RHA1.

Draft Genome Sequence of Comamonas sp. Strain E6 (NBRC 107749), a Degrader of Phthalate Isomers through the Protocatechuate 4,5-Cleavage Pathway.

Comamonas sp. strain E6 can degrade o-phthalate, terephthalate, and isophthalate via the protocatechuate 4,5-cleavage pathway. Here, we report the draft genome sequence of E6 in order to provide insights into its mechanisms in o-phthalate catabolism and its potential use for biotechnological applications.

Complete Genome Sequence of a Dimethyl Sulfide-Utilizing Bacterium, Acinetobacter guillouiae Strain 20B (NBRC 110550).

Acinetobacter guillouiae strain 20B can utilize dimethyl sulfide (DMS) as the sole sulfur source and degrade chloroethylenes. We report here the complete 4,648,418-bp genome sequence for this strain, which contains 4,367 predicted coding sequences (CDSs), including a well-characterized DMS degradative operon.

Gene cluster and regulation system for 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (DDE) degradation in Janibacter sp. TYM3221.

A DDE-degrading bacterium, Janibacter sp. TYM3221, is able to grow on biphenyl and degrades 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (DDE) via a meta-ring cleavage pathway. The bphAa gene, encoding a biphenyl dioxygenase large subunit, was previously demonstrated to be involved in the degradation of DDE in TYM3221. The bph gene cluster, containing orf2 and bphDAaAbAcAdBCST was cloned and characterized. Reverse transcription-PCR (RT-PCR) analysis indicated that these genes were transcribed as an operon. The real-time RT-PCR on orf2, bphAa, bphC, and bphS suggest the presence of the inducible orf2 promoter (orf2p) and constitutive bphAa promoter (bphAap). The TYM3221 bphST conducted biphenyl-dependent inducible activation plus constitutive basal activation of orf2p and constitutive activation of bphAap in a rhodococcal host strain, Rhodococcus erythropolis IAM1399, suggesting that expression of the TYM3221 bph operon depends on the bphST-coded two-component regulatory system. Both of these promoters were also induced by the bphS1T1 of a biphenyl degrader, Rhodococcus jostii RHA1, and contained the 24-bp consensus sequences of RHA1 bphS1T1-dependent promoters. The replacement of RHA1 bphS1 with TYM3221 bphS in combination with RHA1 bphT1 suggests that TYM3221 bphS is responsible for low inducible and high constitutive activation of orf2p in IAM1399 by the TYM3221 bphST-system. Expression of bphAaAbAcAdBC in IAM1399 resulted in the transformation of DDE to the meta-ring cleavage product via 2,3-hydroxylation, suggesting that these genes are involved in DDE degradation.

The 24-bp consensus sequence responsible for regulation of the BphS1T1 two-component system in a hybrid promoter.

Rhodococcus jostii RHA1 degrades polychlorinated biphenyls (PCBs) by cometabolism with biphenyl. The bphS1T1-coding two-component system, which is composed of a sensor kinase, BphS1, and a response regulator, BphT1, activates the transcription of biphenyl/PCB degradation genes from the five promoters of bphAa, etbAa1, etbAa2, etbAd, and etbD1 in the presence of aromatics, such as biphenyl and ethylbenzene. The transcription start sites of etbAd and etbD1 were determined and the results indicated that the 18-bp consensus sequence is shared by all five promoters at the equivalent position from their transcriptional start sites. To investigate the involvement of the 18-bp consensus sequence in the regulation of BphS1T1, a hybrid promoter was constructed by connecting the 18-bp consensus sequence of bphAa promoter to a portion of the benzoate dioxygenase gene promoter, which is not under the control of BphS1T1. The ethylbenzene-dependent induction of the hybrid promoter by BphS1T1 was not observed. Recently, a 24-bp consensus sequence that included the 18-bp consensus sequence of the bphAa promoter was identified in the regions conserved among RHA1 and other rhodococcal degraders. When the 24-bp consensus sequence was employed instead, both BphS1T1-dependent basal activation and ethylbenzene-dependent induction of the hybrid promoter were observed. Mutations in the six extra residues outside the 18-bp sequence in the 24-bp consensus sequence, affected not only ethylbenzene-dependent induction but also BphS1T1-dependent basal activation. The outstanding conservation of the 24-bp consensus sequence was confirmed by multiple sequence alignment. These results indicate that the 24-bp consensus sequence is really responsible for the regulation of BphS1T1.

Dual two-component regulatory systems are involved in aromatic compound degradation in a polychlorinated-biphenyl degrader, Rhodococcus jostii RHA1.

A Gram-positive polychlorinated-biphenyl (PCB) degrader, Rhodococcus jostii RHA1, degrades PCBs by cometabolism with biphenyl. A two-component BphS1T1 system encoded by bphS1 and bphT1 (formerly bphS and bphT) is responsible for the transcription induction of the five gene clusters, bphAaAbAcAdC1B1, etbAa1Ab1CbphD1, etbAa2Ab2AcD2, etbAdbphB2, and etbD1, which constitute multiple enzyme systems for biphenyl/PCB degradation. The bphS2 and bphT2 genes, which encode BphS2 and BphT2, virtually identical to BphS1 (92%) and BphT1 (97%), respectively, were characterized. BphS2T2 induced the activation of the bphAa promoter in a host, Rhodococcus erythropolis IAM1399, in the presence of a variety of aromatics, including benzene, toluene, ethylbenzene, xylenes, isopropylbenzene, and chlorinated benzenes, as effectively as BphS1T1. The substrate spectrum of BphS2T2 was the same as that of BphS1T1, except for biphenyl, which is a substrate only for BphS1T1. BphS2T2 activated transcription from the five promoters of biphenyl/PCB degradation enzyme gene clusters as effectively as BphS1T1. The targeted disruptions of the bphS1, bphS2, bphT1, and bphT2 genes indicated that all these genes are involved in the growth of RHA1 on aromatic compounds. The hybrid system with bphS1 and bphT2 and that with bphS2 and bphT1 were constructed, and both systems conducted induced activation of the bphAa promoter, indicating cross-communication. These results indicated that RHA1 employs not only multiple enzyme systems, but also dual regulatory systems for biphenyl/PCB degradation. Comparison of the sequences, including bphS2T2, with the bphS1T1-containing sequences and the corresponding sequences in other rhodococcal degraders suggests that bphS2T2 might have originated from bphS1T1.

Detection of bphAa gene expression of Rhodococcus sp. strain RHA1 in soil using a new method of RNA preparation from soil.

To understand the response of soil bacteria to the surrounding environment, it is necessary to examine the gene expression profiles of the bacteria in the soil. For this purpose, we developed a new method of extracting RNA from soil reproducibly. Using this new method, we extracted RNA from a field soil, which was sterilized and inoculated with Rhodococcus sp. strain RHA1, a biphenyl degrader isolated from gamma-hexachlorocyclohexane-contaminated soil. Data from agarose gel electrophoresis indicated that the extracted RNA was purified properly. This new method can be applied easily in the preparation of large amounts of RNA. Real-time reverse transcription-polymerase chain reaction (RT-PCR) experiments performed by the TaqMan method suggested that the bphAa gene in this strain, which is involved in the degradation of biphenyl, was induced in the biphenyl amended soil.