Tewes Tralau

Structural studies on the full-length LysR-type regulator TsaR from Comamonas testosteroni T-2 reveal a novel open conformation of the tetrameric LTTR fold

LysR‐type transcriptional regulators (LTTRs) constitute the largest family of regulators in prokaryotes. The full‐length structures of the LTTR TsaR from Comamonas testosteroni T‐2 and its complex with the natural inducer para‐toluensulfonate have been characterized by X‐ray diffraction. Both ligand‐free and complexed forms reveal a dramatically different quaternary structure from that of CbnR from Ralstonia eutropha, or a putative LysR‐type regulator from Pseudomonas aeruginosa, the only other determined full‐length structures of tetrameric LTTRs. Although all three show a head‐to‐head tetrameric ring, TsaR displays an open conformation, whereas CbnR and PA01‐PR present additional contacts in opposing C‐terminal domains that close the ring. Such large differences may be due to a broader structural versatility than previously assumed or either, reflect the intrinsic flexibility of tetrameric LTTRs. On the grounds of the sliding dimer hypothesis of LTTR activation, we propose a structural model in which the closed structures could reflect the conformation of a ligand‐free LTTR, whereas inducer binding would bring about local changes to disrupt the interface linking the two compact C‐terminal domains. This could lead to a TsaR‐like, open structure, where the pairs of recognition helices are closer to each other by more than 10u2003Å.

Map of the IncP1β Plasmid pTSA Encoding the Widespread Genes (tsa) for p-Toluenesulfonate Degradation in Comamonas testosteroni T-2

ABSTRACT The catabolic IncP1β plasmid pTSA from Comamonas testosteroni T-2 was mapped by subtractive analysis of restriction digests, by sequencing outwards from the tsaoperon (toluenesulfonate degradation), and by generating overlapping, long-distance-PCR amplification products. The plasmid was estimated to comprise 72 ± 4 kb. The tsa region was found to be a composite transposon flanked by two IS1071 elements. A cryptic tsa operon was also present in the tsa transposon. Those backbone genes and regions which we sequenced were in the same order as the corresponding genes in resistance plasmid R751, and identities of about 99% were observed. Enrichment cultures with samples from four continents were done to obtain organisms able to utilizep-toluenesulfonate as the sole source of carbon and energy for aerobic growth. Most (15) of the 16 cultures (13 of them isolates) were obtained from contaminated sites and were attributed to three metabolic groups, depending on their metabolism ofp-toluenesulfonate. The largest group contained thetsa transposon, usually (six of seven isolates) with negligible differences in sequence from strain T-2.

Transcriptomic Analysis of the Sulfate Starvation Response of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen that causes a number of infections in humans, but is best known for its association with cystic fibrosis. It is able to use a wide range of sulfur compounds as sources of sulfur for growth. Gene expression in response to changes in sulfur supply was studied in P. aeruginosa E601, a cystic fibrosis isolate that displays mucin sulfatase activity, and in P. aeruginosa PAO1. A large family of genes was found to be upregulated by sulfate limitation in both isolates, encoding sulfatases and sulfonatases, transport systems, oxidative stress proteins, and a sulfate-regulated TonB/ExbBD complex. These genes were localized in five distinct islands on the genome and encoded proteins with a significantly reduced content of cysteine and methionine. Growth of P. aeruginosa E601 with mucin as the sulfur source led not only to a sulfate starvation response but also to induction of genes involved with type III secretion systems.

A novel outer-membrane anion channel (porin) as part of a putatively two-component transport system for 4-toluenesulphonate in Comamonas testosteroni T-2

Inducible mineralization of TSA (4-toluenesulphonate) by Comamonas testosteroni T-2 is initiated by a secondary transport system, followed by oxygenation and oxidation by TsaMBCD to 4-sulphobenzoate under the regulation of TsaR and TsaQ. Evidence is presented for a novel, presumably two-component transport system (TsaST). It is proposed that TsaT, an outer-membrane porin, formed an anion-selective channel that works in co-operation with the putative secondary transporter, TsaS, located in the inner membrane. tsaT was identified as a 1017-bp ORF (open reading frame) on plasmid pTSA upstream of the TSA-catabolic genes in the tsa operon. Expression of tsaT was regulated by TsaR, the transcriptional activator of the tsa regulon. The presence of tsaT was concomitant with the presence of the tsa operon in different TSA-degrading isolates. tsaT was expressed in Escherichia coli and was detected in the outer membrane. A 22-amino-acid leader peptide was identified. Purified protein reconstituted in lipid bilayer membranes formed anion-selective channels with a single-channel conductance of 3.5 nS in 1 M KCl. Downstream of tsaT, a constitutively expressed 720-bp ORF (tsaS) was identified. tsaS coded for a hydrophobic protein predicted to have six transmembrane helices and which is most likely localized in the cytoplasmic membrane. tsaS is adjacent to tsaT, but showed a different transcriptional profile.

Degradation of benzo[a]pyrene by bacterial isolates from human skin

Polycyclic aromatic hydrocarbons (PAHs) are some of the most widespread xenobiotic pollutants, with the potentially carcinogenic high-molecular-weight representatives being of particular interest. However, while in eukaryotes, the cytochrome P450 (CYP)-mediated activation of benzo[a]pyrene (B[a]P) has become a model for metabolism-mediated carcinogenesis, the oxidative degradation of B[a]P by microorganisms is less well studied. This should be reason for concern as the human organ most exposed to environmental PAHs is the skin, which at the same time is habitat to a most diverse population of microbial commensals. Yet, nothing is known about the skins microbiome potential to metabolise B[a]P. This study now reports on the isolation of 21 B[a]P-degrading microorganisms from human skin, 10 of which were characterised further. All isolates were able to degrade B[a]P as sole source of carbon and energy, and degradation was found to be complete in at least four isolates. Substrate metabolism involved two transcripts that encode a putative DszA/NtaA-like monooxygenase and a NifH-like reductase, respectively. Analysis of the 16S-rRNA genes showed that the B[a]P-degrading isolates comprise Gram(+) as well as Gram(-) skin commensals, with Micrococci being predominant. Moreover, microbial B[a]P-degradation was detected on all volunteers probed, indicating it to be a universal feature of the skins microbiome.

Characterization of TsaR, an Oxygen-Sensitive LysR-Type Regulator for the Degradation of p-Toluenesulfonate in Comamonas testosteroni T-2

ABSTRACT TsaR is the putative LysR-type regulator of the tsa operon (tsaMBCD) which encodes the first steps in the degradation of p-toluenesulfonate (TSA) in Comamonas testosteroni T-2. Transposon mutagenesis was used to knock out tsaR. The resulting mutant lacked the ability to grow with TSA and p-toluenecarboxylate (TCA). Reintroduction of tsaR in trans on an expression vector reconstituted growth with TSA and TCA. The tsaR gene was cloned into Escherichia coli with a C-terminal His tag and overexpressed as TsaRHis. TsaRHis was subject to reversible inactivation by oxygen, which markedly influenced the experimental approaches used. Gel filtration showed TsaRHis to be a monomer in solution. Overexpressed TsaRHis bound specifically to three regions within the promoter between the divergently transcribed tsaR and tsaMBCD. The dissociation constant (KD) for the whole promoter region was about 0.9 μM, and the interaction was a function of the concentration of the ligand TSA. A regulatory model for this LysR-type regulator is proposed on the basis of these data.

Wind of change challenges toxicological regulators.

Background: In biomedical research, the past two decades have seen the advent of in vitro model systems based on stem cells, humanized cell lines, and engineered organotypic tissues, as well as numerous cellular assays based on primarily established tumor-derived cell lines and their genetically modified derivatives. Objective: There are high hopes that these systems might replace the need for animal testing in regulatory toxicology. However, despite increasing pressure in recent years to reduce animal testing, regulators are still reluctant to adopt in vitro approaches on a large scale. It thus seems appropriate to consider how we could realistically perform regulatory toxicity testing using in vitro assays only. Discussion and Conclusion: Here, we suggest an in vitro–only approach for regulatory testing that will benefit consumers, industry, and regulators alike.

An additional regulator, TsaQ, is involved with TsaR in regulation of transport during the degradation of p-toluenesulfonate in Comamonas testosteroni T-2

Abstract. The degradation of p-toluenesulfonate (TSA) by Comamonas testosteroni T-2 is initiated by a transport system (TsaST) and enzymes (TsaMBCD) encoded on the tsa transposon, Tntsa, on the TSA plasmid (pTSA). Tntsa comprises an insert of 15xa0kb between two IS1071 elements. The left-hand 6xa0kb and the right-hand 6xa0kb are nearly mirror images. The regulator of the tsaMBCD1 genes (right-hand side) is the centrally located LysR-type TsaR, which is encoded upstream of tsaMBCD1 on the reverse strand. The other centrally located genes are tsaS and tsaT, encoded downstream of tsaR and on the same strand as both tsaR and tsaMBCD2. The latter four genes are not expressed. Downstream of tsaD1 (tsaD2) is tsaQ1 (tsaQ2) and another open reading frame of unknown function. The tsaQ genes have identical sequences. Sequence analysis indicated that TsaQ could be an IclR-type regulator, whose expression during degradation of TSA was proven by data from RT-PCR. Both copies of tsaQ could be knocked-out by homologous recombination. Double mutants failed to grow with TSA but grew with p-toluenecarboxylate (TCA), which is also degraded via TsaMBCD. This showed TsaQ to be essential for the degradation of TSA but not TCA. We attributed this to regulation of the transport of TSA, especially to regulation of the expression of tsaT, which was expressed solely during growth with TSA. Seven independently isolated bacteria containing the tsa operon were available. Those six which contained tsaT on Tntsa also contained tsaQ. The promoter region of tsaT was found to be a target of the regulator TsaR. Band-shift data indicate that TsaR is required for the expression of tsaT, which suggests that tsaR and tsaQ1,2, together with tsaMBCD1, belong to a common regulatory unit.

Chemical toxicity testing in vitro using cytochrome P450-expressing cell lines, such as human CYP1B1

This protocol describes how to use cytochrome P450–dependent monooxygenase (CYP)-expressing cell lines in toxicity testing of chemicals in vitro. Selected cells amenable to permanently grow in culture are genetically manipulated to stably express single CYP enzymes originating from any species of interest. This expression can be characterized by, for example, determining CYP mRNA content, CYP protein level (western blotting or in situ immunofluorescence) and CYP-mediated enzyme activity (substrate conversion assays). These cells can be used to determine substrate specificities and species differences, e.g., in the bioactivation of drugs. Once constructed, CYP-expressing cells can serve as a straightforward and reliable tool in toxicity testing and the corresponding assays could be adapted for high-throughput analysis. Using these cells, enzyme assays can be performed in a matter of hours. This protocol is exemplified with V79 fibroblasts from Chinese hamster (Cricetulus griseus), modified to express human cytochrome P450 1B1 (CYP1B1). These cells are characterized for their CYP1B1-linked properties by in situ immunofluorescence and their activity in the 7-ethoxyresorufin-O-deethylase enzyme assay. This is followed by an assay showing metabolic activation of the polycyclic aromatic hydrocarbon dibenzo[a,l]pyrene by CYP1B1, along with the toxicological endpoints of cytotoxicity and micronucleus formation.

Why two are not enough : degradation of p-toluenesulfonate by a bacterial community from a pristine site in Moorea, French Polynesia

In previous work, only one culture (strain TA12) from a pristine site was reported to utilize the xenobiotic compound p-toluenesulfonate (TSA) as a sole source of carbon and energy for aerobic growth. Strain TA12 has now been recognized as a community of three bacteria: Achromobacter xylosoxidans TA12-A, Ensifer adhaerens TA12-B and Pseudomonas nitroreducens TA12-C. Achromobacter xylosoxidans TA12-A and E. adhaerens TA12-B were identified as the TSA degraders. These two organisms contain several tsa genes from the Tntsa cluster described previously in Comamonas testosteroni T-2 and use the tsa pathway. Apparently, due to vitamin auxotrophy, the growth of the pure cultures with TSA was markedly slower than the growth of the community with TSA. The third bacterium (P. nitroreducens) TA12-C is, then, a provider of essential vitamins for the TSA degraders and occurs at a low frequency.