mfsQ encoding an MFS efflux pump mediates adaptive protection of Stenotrophomonas maltophilia against benzalkonium chloride

The persistence of Stenotrophomonas maltophilia, especially in hospital environments where disinfectants are used intensively, is one of the important factors that allow this opportunistic pathogen to establish nosocomial infections. In the present study, we illustrated that S. maltophilia possesses adaptive resistance to the disinfectant benzalkonium chloride (BAC). This BAC adaptation was abolished in the ΔmfsQ mutant, in which a gene encoding an efflux transporter belonging to the major facilitator superfamily (MFS) was deleted. The ΔmfsQ mutant also showed increased susceptibility to BAC and chlorhexidine gluconate relative to a parental wild type. The expression of mfsQ increased upon exposure to quaternary ammonium compounds, including BAC. Thus, the results of this study suggest that mfsQ plays a role in both adaptive and non-adaptive protection of S. maltophilia from the toxicity of the disinfectant BAC.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Agrobacterium tumefaciens soxR Is Involved in Superoxide Stress Protection and Also Directly Regulates Superoxide-Inducible Expression of Itself and a Target Gene

Inactivation of Agrobacterium tumefaciens soxR increases sensitivity to superoxide generators. soxR expression is highly induced by superoxide stress and is autoregulated. SoxR also directly regulates the superoxide-inducible expression of atu5152. Taken together, the physiological role of soxR and the mechanism by which it regulates expression of target genes make the A. tumefaciens SoxR system different from other bacterial systems

Agrobacterium tumefaciens estC, Encoding an Enzyme Containing Esterase Activity, Is Regulated by EstR, a Regulator in the MarR Family.

Analysis of the A. tumefaciens genome revealed estC, which encodes an esterase located next to its transcriptional regulator estR, a regulator of esterase in the MarR family. Inactivation of estC results in a small increase in the resistance to organic hydroperoxides, whereas a high level of expression of estC from an expression vector leads to a reduction in the resistance to organic hydroperoxides and menadione. The estC gene is transcribed divergently from its regulator, estR. Expression analysis showed that only high concentrations of cumene hydroperoxide (CHP, 1 mM) induced expression of both genes in an EstR-dependent manner. The EstR protein acts as a CHP sensor and a transcriptional repressor of both genes. EstR specifically binds to the operator sites OI and OII overlapping the promoter elements of estC and estR. This binding is responsible for transcription repression of both genes. Exposure to organic hydroperoxide results in oxidation of the sensing cysteine (Cys16) residue of EstR, leading to a release of the oxidized repressor from the operator sites, thereby allowing transcription and high levels of expression of both genes. The estC is the first organic hydroperoxide-inducible esterase-encoding gene in alphaproteobacteria

Optimization of Cyclodextrin Production From Sago Starch

Cyclodextrin (CD) is synthesized by bacterial cyclodextrin glycosyltransferase (CGTase) and is widely used in food, pharmaceutical, cosmetic, and agricultural industries. In this study, Bacillus circulans CGTase was partially purified by ammonium sulfate precipitation at 50--70% saturation. The optimum pH and temperature for CD production from sago starch were found to be in the ranges of 4.5--5.0 and 55--60 #C, respectively. b-CD was the predominant product, constituting 65% of all CD products. The b-CD produced using partially purified and crude CGTase were compared and found to have no significant di#erence in yield and productivity. The appropriate proportion of CGTase to sago starch for b-CD production was determined by response surface methodology. The most appropriate enzyme:substrate ratio was 50 U g sago starch #1 CGTase and 60 g l #1 sago starch

Multiple alignments of Atu5211 (EstR) and Atu5212 (EstC).

<p>A, The deduced amino acid sequence of Atu5211 (EstR) was aligned with OhrR from <i>Xanthomonas campestris</i> pv. phaseoli (OhrR_Xcp), <i>A</i>. <i>tumefaciens</i> (OhrR_Atu) and <i>Bacillus subtilis</i> (OhrR_Bsu). Cysteine residues are marked by arrow heads. B, A phylogenetic tree constructed from the amino acid sequences of transcriptional regulators belonging to MarR super family. EstR_Atu, <i>A</i>. <i>tumefaciens</i> EstR; OhrR_Xcp, <i>Xanthomonas campestris</i> pv. phaseoli OhrR (AAK62673); OhrR_Bsu, <i>Bacillus subtilis</i> OhrR (NP_389198); OhrR_Sco, <i>Streptomyces coelicolor</i> OhrR (CAB87337); OhrR_Atu, <i>Agrobacterium tumefaciens</i> OhrR (AAK86653); ExpG_Sme, <i>Sinorhizobium meliloti</i> ExpG (CAB01941); MepR_Sau, <i>Staphylococcus aureus</i> MepR (AAU95767), GraR_Rhi, <i>Rhizobium sp</i> GraR (BAF44528); TcaR_Sau, <i>Staphylococcus aureus</i> TcaR (AAG23887); ScoC_Bsu, <i>Bacillus subtilis</i> ScoC (NP_388880); PecS_Ech, <i>Erwinia chrysanthemi</i> PecS (CAA52427); HucR_Dra, <i>Deinococcus radiodurans</i> HucR (NP_294883); MexR_Pae, <i>Pseudomonas aeruginosa</i> MexR (AAO40258); MarR_Eco, <i>Escherichia coli</i> MarR (ABE11597); SlyA_Sty, <i>Salmonella typhimurium</i> SlyA (NP_460407); SlyA_Efa, <i>Enterococcus faecalis</i> SlyA (NP_816617); MobR_Cte, <i>Comamonas testosterone</i> MobR (BAF34929); BadR_Rpa, <i>Rhodopseudomonas palustris</i> BadR (NP_946008); MoaI_Kae, <i>Klebsiella aerogenes</i> MoaI (BAA09790); and HpcR_Eco, <i>Escherichia coli</i> HpcR (AAB25801). C, The deduced amino acid sequence of Atu5212 (EstC) was aligned with EstC from <i>Burkholderia gladioli</i> EstC [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168791#pone.0168791.ref038" target="_blank">38</a>]. The Ser-His-Asp catalytic triad residues are indicated by asterisks. The bar above the sequences represents the esterase-lipase active domain. Alignment was performed using ClustalW [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168791#pone.0168791.ref032" target="_blank">32</a>].</p

Regulation by SoxR of mfsA, Which Encodes a Major Facilitator Protein Involved in Paraquat Resistance in Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia MfsA (Smlt1083) is an efflux pump in the major facilitator superfamily (MFS). Deletion of mfsA renders the strain more susceptible to paraquat, but no alteration in the susceptibility levels of other oxidants is observed. The expression of mfsA is inducible upon challenge with redox cycling/superoxide-generating drug (paraquat, menadione and plumbagin) treatments and is directly regulated by SoxR, which is a transcription regulator and sensor of superoxide-generating agents. Analysis of mfsA expression patterns in wild-type and a soxR mutant suggests that oxidized SoxR functions as a transcription activator of the gene. soxR (smlt1084) is located in a head-to-head fashion with mfsA, and these genes share the -10 motif of their promoter sequences. Purified SoxR specifically binds to the putative mfsA promoter motifs that contain a region that is highly homologous to the consensus SoxR binding site, and mutation of the SoxR binding site abolishes binding of purified SoxR protein. The SoxR box is located between the putative -35 and -10 promoter motifs of mfsA; and this position is typical for a promoter in which SoxR acts as a transcriptional activator. At the soxR promoter, the SoxR binding site covers the transcription start site of the soxR transcript; thus, binding of SoxR auto-represses its own transcription. Taken together, our results reveal for the first time that mfsA is a novel member of the SoxR regulon and that SoxR binds and directly regulates its expression

Gene organization of the <i>estC</i> locus in various bacteria.

<p>The arrow indicates the orientation of the transcription. The number in the arrows represents percentage of identity of each sequence to the corresponding <i>A</i>. <i>tumefaciens</i> genes. The asterisk indicates a truncated gene. <i>scd</i>, short chain dehydrogenase; <i>zdh</i>, zinc dehydrogenase; <i>cdh</i>, choline dehydrogenase; and <i>gst</i>, glutathione S-transferase.</p

Expression analysis of <i>estC</i>.

<p>A, Northern analysis of <i>estC</i> in <i>A</i>. <i>tumefaciens</i> NTL4 wild-type and the <i>estR</i> mutant was performed using the [³²P]-labeled <i>estC</i> probe. Equal amounts of RNA (10 μg) prepared from bacterial cultures grown under uninduced (UN) conditions or induced with 1 mM CHP, 250 μM H₂O₂, 200 μM MD or 250 μM plumbagin (PB) were used in the experiments. The 23S rRNA used as the amount control is shown below the hybridized autoradiograph. B, Esterase activity in <i>A</i>. <i>tumefaciens</i> NTL4 and the <i>estR</i> mutant strains was assayed in crude lysates prepared from cultures cultivated as described in A. BHP, culture induced with 500 μM <i>t</i>-butyl hydroperoxide. C and D show the <i>estC</i> and <i>estR</i> promoter activities <i>in vivo</i>, respectively, in NTL4, the <i>estR</i> mutant, and the complemented strain (<i>estR</i>/pEstR) carrying the plasmid either pP_estC that contains the <i>estC</i> promoter-<i>lacZ</i> fusion (C) or pP_estR that contains the <i>estR</i> promoter-<i>lacZ</i> fusion (D). The activities were monitored in samples of lysates prepared from uninduced (UN) and oxidant-induced cultures (1 mM CHP, 25 μM LHP, 500 μM BHP, 250 μM H₂O₂, and 250 μM MD). The β-galactosidase activity is expressed in international units. pEstR means harboring the <i>estR</i> expression plasmid. Asterisks indicate a significant difference (<i>P</i> < 0.05) from the uninduced control.</p