Salmonella enterica synthesizes 5,6‐dimethylbenzimidazolyl‐(DMB)‐α‐riboside. Why some Firmicutes do not require the canonical DMB activation system to synthesize adenosylcobalamin

5,6‐Dimethylbenzimidazolyl‐(DMB)‐ α ‐ribotide [ α ‐ribazole‐5′‐phosphate ( α ‐RP)] is an intermediate in the biosynthesis of adenosylcobalamin (AdoCbl) in many prokaryotes. In such microbes, α ‐RP is synthesized by nicotinate mononucleotide (NaMN):DMB phosphoribosyltransferases (CobT in Salmonella enterica), in a reaction that is considered to be the canonical step for the activation of the base of the nucleotide present in adenosylcobamides. Some Firmicutes lack CobT‐type enzymes but have a two‐protein system comprised of a transporter (i.e., CblT) and a kinase (i.e., CblS) that can salvage exogenous α ‐ribazole ( α ‐R) from the environment using CblT to take up α ‐R, followed by α ‐R phosphorylation by CblS. We report that Geobacillus kaustophilus CblT and CblS proteins restore α ‐RP synthesis in S. enterica lacking the CobT enzyme. We also show that a S. enterica cobT strain that synthesizes GkCblS ectopically makes only AdoCbl, even under growth conditions where the synthesis of pseudoCbl is favored. Our results indicate that S. enterica synthesizes α ‐R, a metabolite that had not been detected in this bacterium and that GkCblS has a strong preference for DMB‐ribose over adenine‐ribose as substrate. We propose that in some Firmicutes DMB is activated to α ‐RP via α ‐R using an as‐yet‐unknown route to convert DMB to α ‐R and CblS to convert α ‐R to α ‐RP.     

A new pathway for the synthesis of α‐ribazole‐phosphate in Listeria innocua

The genomes of Listeria spp. encode all but one of 25 enzymes required for the biosynthesis of adenosylcobalamin (AdoCbl; coenzyme B₁₂). Notably, all Listeria genomes lack CobT, the nicotinamide mononucleotide:5,6‐dimethylbenzimidazole (DMB) phosphoribosyltransferase (EC 2.4.2.21) enzyme that synthesizes the unique α‐linked nucleotide N¹‐(5‐phospho‐α‐d ‐ribosyl)‐DMB (α‐ribazole‐5′‐P, α‐RP), a precursor of AdoCbl. We have uncovered a new pathway for the synthesis of α‐RP in Listeria innocua that circumvents the lack of CobT. The cblT and cblS genes (locus tags lin1153 and lin1110) of L. innocua encode an α‐ribazole (α‐R) transporter and an α‐R kinase respectively. Results from in vivo experiments indicate that L. innocua depends on CblT and CblS activities to salvage exogenous α‐R, allowing conversion of the incomplete corrinoid cobinamide (Cbi) into AdoCbl. Expression of the L. innocua cblT and cblS genes restored AdoCbl synthesis from Cbi and α‐R in a Salmonella enterica cobT strain. LinCblT transported α‐R across the cell membrane, but not α‐RP or DMB. UV‐visible spectroscopy and mass spectrometry data identified α‐RP as the product of the ATP‐dependent α‐R kinase activity of LinCblS. Bioinformatics analyses suggest that α‐R salvaging occurs in important Gram‐positive human pathogens.     

Structural investigation of the biosynthesis of alternative lower ligands for cobamides by nicotinate mononucleotide: 5,6-dimethylbenzimidazole phosphoribosyltransferase from Salmonella enterica.

Nicotinate mononucleotide (NaMN):5,6-dimethylbenzimidazole phosphoribosyltransferase (CobT) from Salmonella enterica plays a central role in the synthesis of alpha-ribazole, a key component of the lower ligand of cobalamin. Surprisingly, CobT can phosphoribosylate a wide range of aromatic substrates, giving rise to a wide variety of lower ligands in cobamides. To understand the molecular basis for this lack of substrate specificity, the x-ray structures of CobT complexed with adenine, 5-methylbenzimidazole, 5-methoxybenzimidazole, p-cresol, and phenol were determined. Furthermore, adenine, 5-methylbenzimidazole, 5-methoxybenzimidazole, and 2-hydroxypurine were observed to react with NaMN within the crystal lattice and undergo the phosphoribosyl transfer reaction to form product. Significantly, the stereochemistries of all products are identical to those found in vivo. Interestingly, p-cresol and phenol, which are the lower ligand in Sporomusa ovata, bound to CobT but did not react with NaMN. This study provides a structural explanation for how CobT can phosphoribosylate most of the commonly observed lower ligands found in cobamides with the exception of the phenolic lower ligands observed in S. ovata. This is accomplished with minor conformational changes in the side chains that constitute the 5,6-dimethylbenzimidazole binding site. These investigations are consistent with the implication that the nature of the lower ligand is controlled by metabolic factors rather by the specificity of the phosphoribosyltransferase.     

ArsD: an As(III) metallochaperone for the ArsAB As(III)-translocating ATPase

The toxic metalloid arsenic is widely disseminated in the environment and causes a variety of health and environment problems. As an adaptation to arsenic-contaminated environments, organisms have developed resistance systems. Many ars operons contain only three genes, arsRBC. Five gene ars operons have two additional genes, arsD and arsA, and these two genes are usually adjacent to each other. ArsA from Escherichia coli plasmid R773 is an ATPase that is the catalytic subunit of the ArsAB As(III) extrusion pump. ArsD was recently identified as an arsenic chaperone to the ArsAB pump, transferring the trivalent metalloids As(III) and Sb(III) to the ArsA subunit of the pump. This increases the affinity of ArsA for As(III), resulting in increased rates if extrusion and resistance to environmentally relevant concentrations of arsenite. ArsD is a homodimer with three vicinal cysteine pairs, Cys12–Cys13, Cys112–Cys113 and Cys119–Cys120, in each subunit. Each vicinal pair binds one As(III) or Sb(III). ArsD mutants with alanines substituting for Cys112, Cys113, Cys119 or Cys120, individually or in pairs or truncations lacking the vicinal pairs, retained ability to interact with ArsA, to activate its ATPase activity. Cells expressing these mutants retained ArsD-enhanced As(III) efflux and resistance. In contrast, mutants with substitutions of conserved Cys12, Cys13 or Cys18, individually or in pairs, were unable to activate ArsA or to enhance the activity of the ArsAB pump. It is proposed that ArsD residues Cys12, Cys13 and Cys18, but not Cys112, Cys113, Cys119 or Cys120, are required for delivery of As(III) to and activation of the ArsAB pump.     

Functional Analysis of the Nicotinate Mononucleotide:5,6-Dimethylbenzimidazole Phosphoribosyltransferase (CobT) Enzyme,Involved in the Late Steps of Coenzyme B12 Biosynthesis in Salmonella enterica

In Salmonella enterica, the CobT enzyme activates the lower ligand base during the assembly of the nucleotide loop of adenosylcobalamin (AdoCbl) and other cobamides. Previously, mutational analysis identified a class of alleles (class M) that failed to restore AdoCbl biosynthesis during intragenic complementation studies. To learn why class M cobT mutations were deleterious, we determined the nature of three class M cobT alleles and performed in vivo and in vitro functional analyses guided by available structural data on the wild-type CobT (CobT^WT) enzyme. We analyzed the effects of the variants CobT(G257D), CobT(G171D), CobT(G320D), and CobT(C160A). The latter was not a class M variant but was of interest because of the potential role of a disulfide bond between residues C160 and C256 in CobT activity. Substitutions G171D, G257D, and G320D had profound negative effects on the catalytic efficiency of the enzyme. The C160A substitution rendered the enzyme fivefold less efficient than CobT^WT. The CobT(G320D) protein was unstable, and results of structure-guided site-directed mutagenesis suggest that either variants CobT(G257D) and CobT(G171D) have less affinity for 5,6-dimethylbenzimidazole (DMB) or access of DMB to the active site is restricted in these variant proteins. The reported lack of intragenic complementation among class M cobT alleles is caused in some cases by unstable proteins, and in others it may be caused by the formation of dimers between two mutant CobT proteins with residual activity that is so low that the resulting CobT dimer cannot synthesize sufficient product to keep up with even the lowest demand for AdoCbl.Cobalamin (Cbl, also known as B₁₂) is a structurally complex cyclic tetrapyrrole with a cobalt ion coordinated by equatorial bonds with pyrrolic nitrogen atoms and is unique among cyclic tetrapyrroles (e.g., heme, chlorophylls, coenzyme F₄₃₀) in that it has an upper and a lower axial ligand (Fig. ​(Fig.1).1). The coenzymic form of Cbl is known as adenosylcobalamin (AdoCbl) or coenzyme B₁₂.Open in a separate windowFIG. 1.Role of CobT in the late steps of AdoCbl biosynthesis. This branch of the AdoCbl biosynthetic pathway is known as the NLA pathway. The black box in the inner membrane represents the corrinoid-specific ABC transporter BtuCD. The inset shows the chemical structure of AdoCbl; the coring ring in the scheme is represented by the rhomboid cartoon with the Co ion in the middle.Some bacteria and archaea synthesize AdoCbl de novo or from preformed precursors such as cobyric acid (Cby) or cobinamide (Cbi) (Fig. ​(Fig.1)1) (11, 32). The enterobacterium Salmonella enterica serovar Typhimurium LT2 (hereafter referred to as S. Typhimurium) synthesizes the corrin ring of AdoCbl de novo only under anoxic conditions (15). Although oxygen blocks de novo corrin ring biosynthesis in this bacterium, it does not block the assembly of adenosylcobalamin (AdoCbl, coenzyme B₁₂) if cells are provided with preformed, incomplete corrinoids such as Cbi or Cby (11, 13-15).The late steps in AdoCbl biosynthesis can be divided into two different branches that comprise the nucleotide loop assembly (NLA) pathway (19). One of the branches of the NLA pathway activates the lower ligand base, while the other one activates adenosylcobinamide (AdoCbi) to AdoCbi-GDP (Fig. ​(Fig.11).In this paper, we focus on the activation of 5,6-dimethylbenzimidazole (DMB), the lower ligand base of AdoCbl. There are two ways in which DMB can be activated. In both cases, the CobT enzyme (EC 2.4.2.21) catalyzes the reaction, but the product of the reaction varies, depending on whether the cosubstrate of CobT is NAD⁺ or its precursor nicotinate mononucleotide (NaMN). If NaMN is the substrate, CobT synthesizes α-ribazole-phosphate (α-RP) (reaction: DMB + NaMN → α-RP + Na) (30). If NAD⁺ substitutes for NaMN, CobT synthesizes α-DMB adenine dinucleotide (α-DAD) (reaction: DMB + NAD⁺ → α-DAD + Nm) (18). α-RP is a good substrate for the AdoCbl-5′-phosphate (AdoCbl-P) synthase (CobS; EC 2.7.8.26) enzyme (19, 34), suggesting that an unidentified enzyme cleaves α-DAD into α-RP and AMP (reaction: α-DAD → α-RP + AMP). Although it is possible that CobS may use α-DAD as a substrate, to date, data are not available to support this idea. The AdoCbl-P phosphatase (CobC; EC 3.1.3.73) enzyme catalyzes the last step of the NLA pathway to yield AdoCbl (Fig. ​(Fig.1)1) (34).Early genetic studies identified four classes of cobT alleles, namely, classes J, K, L, and M (12); class M was an intriguing class of mutations because they did not display intragenic complementation (12). Here we identify the nature of class M cobT mutations, report the initial in vitro and in vivo characterization of class M CobT variant proteins, and propose structural explanations for the observed deficiencies in CobT activity caused by class M mutations.     

Cloning of Petunia hybrida chloroplast DNA sequences capable of autonomous replication in yeast

Summary Sequences from Petunia hybrida chloroplast DNA which have the property to promote autonomous replication in Saccharomyces cerevisiae were cloned in vector YIp5. Seven cloned chloroplast DNA fragments are localized at one of two different sites on the chloroplast genome. One site, arsA was mapped on a 1.8 Kb fragment at position 27.0–28.8 Kb on the P. hybrida chloroplast genome. The plasmids containing this arsA are stable both in yeast and E. coli. The other site, arsB, was shown to be very unstable and is located either in the small single copy region close to the inverted repeat or just in the inverted repeat. The functioning of these sequences as a possible origin of replication in vivo is discussed.     

Behavioural responses of stable flies to cattle manure slurry associated odourants

Stable flies (Stomoxys calcitrans [Diptera: Muscidae] L.) are blood‐feeding synanthropic pests, which cause significant economic losses in livestock. Stable fly antennae contain olfactory sensilla responsive to host and host environment‐associated odours. Field observation indicated that the abundance of stable flies increased significantly in grasslands or crop fields when cattle manure slurry was applied. Major volatile compounds emanating from manure slurry were collected and identified. Behavioural responses of stable flies to those compounds were investigated in laboratory bioassays and field‐trapping studies. Results from olfactometer assays revealed that phenol, p‐cresol and m‐cresol were attractive to adult stable flies. When tested individually, attraction was higher with lower dosages. Stable flies were most attracted to blends of phenol and m‐cresol or p‐cresol. Traps with binary blend lures caught more stable flies in field trials as well.     

Dissecting cobamide diversity through structural and functional analyses of the base-activating CobT enzyme of Salmonella enterica

Background

Cobamide diversity arises from the nature of the nucleotide base. Nicotinate mononucleotide (NaMN):base phosphoribosyltransferases (CobT) synthesize α-linked riboside monophosphates from diverse nucleotide base substrates (e.g., benzimidazoles, purines, phenolics) that are incorporated into cobamides.

Methods

Structural investigations of two members of the CobT family of enzymes in complex with various substrate bases as well as in vivo and vitro activity analyses of enzyme variants were performed to elucidate the roles of key amino acid residues important for substrate recognition.

Results

Results of in vitro and in vivo studies of active-site variants of the Salmonella enterica CobT (SeCobT) enzyme suggest that a catalytic base may not be required for catalysis. This idea is supported by the analyses of crystal structures that show that two glutamate residues function primarily to maintain an active conformation of the enzyme. In light of these findings, we propose that proper positioning of the substrates in the active site triggers the attack at the C1 ribose of NaMN.

Conclusion

Whether or not a catalytic base is needed for function is discussed within the framework of the in vitro analysis of the enzyme activity. Additionally, structure-guided site-directed mutagenesis of SeCobT broadened its substrate specificity to include phenolic bases, revealing likely evolutionary changes needed to increase cobamide diversity, and further supporting the proposed mechanism for the phosphoribosylation of phenolic substrates.

General Significance

Results of this study uncover key residues in the CobT enzyme that contribute to the diversity of cobamides in nature.     

Isolation of the ATP-Binding Human Homolog of thearsAComponent of the Bacterial Arsenite Transporter

Arsenite resistance in bacteria is mediated by an efflux pump composed of thearsAandarsBgene products. We have isolated the human homolog of the bacterialarsA(hARSA-I), a member of the ATPase superfamily with no transmembrane domain. Southern and Northern analyses indicated the presence of two cross-hybridizing genes in the human genome and expression ofhARSA-Iin many tissues. A rabbit antiserum raised against a glutathione-S-transferase (GST)/hARSA-I fusion protein identified two cross-reacting proteins of 37 and 42 kDa by Western analysis in two different human cell lines. Overexpression ofhARSA-Iin the embryonal human kidney 293 cell line was accompanied by overproduction of the 37-kDa protein. Biochemical analysis using the GST/hARSA-I fusion protein indicated that hARSA-I is an ATPase analogous to the bacterial ArsA. Thus,hARSA-Iis a new eukaryotic member of a highly conserved ATP-binding superfamily of proteins.     

Homology of Escherichia coli R773 arsA, arsB, and arsC Genes in Arsenic-Resistant Bacteria Isolated from Raw Sewage and Arsenic-Enriched Creek Waters

The occurrence and diversity of the Escherichia coli R773 ars operon were investigated among arsenic-resistant enteric and nonenteric bacteria isolated from raw sewage and arsenic-enriched creek waters. Selected isolates from each creek location were screened for ars genes by colony hybridization and PCR. The occurrence of arsA, arsB, and arsC determined by low-stringency colony hybridization (31 to 53% estimated mismatch) was 81, 87, and 86%, respectively, for 84 bacteria isolated on arsenate- and arsenite-amended media from three locations. At moderate stringency (21 to 36% estimated mismatch), the occurrence decreased to 42, 56, and 63% for arsA, arsB, and arsC, respectively. PCR results showed that the ars operon is conserved in some enteric bacteria isolated from creek waters and raw sewage. The occurrence of the arsBC genotype was about 50% in raw sewage enteric bacteria, while arsA was detected in only 9.4% of the isolates (n = 32). The arsABC and arsBC genotypes occurred more frequently in enteric bacteria isolated from creek samples: 71.4 and 85.7% (n = 7), respectively. Average sequence divergence within arsB for six creek enteric bacteria was 20% compared to that of the E. coli R773 ars operon. Only 1 of 11 pseudomonads screened by PCR was positive for arsB. The results from this study suggest that significant divergence has occurred in the ars operon among As-resistant E. coli strains and in Pseudomonas spp.     

The end of the cob operon: evidence that the last gene (cobT) catalyzes synthesis of the lower ligand of vitamin B12, dimethylbenzimidazole.

The cob operon of Salmonella typhimurium includes 20 genes devoted to the synthesis of adenosyl-cobalamin (coenzyme B12). Mutants with lesions in the promoter-distal end of the operon synthesize vitamin B12 only if provided with 5,6-dimethylbenzimidazole (DMB), the lower ligand of vitamin B12. In the hope of identifying a gene(s) involved in synthesis of DMB, the DNA base sequence of the end of the operon has been determined; this completes the sequence of the cob operon. The cobT gene is the last gene in the operon. Four CobII (DMB-) mutations mapping to different deletion intervals of the CobII region were sequenced; all affect the cobT open reading frame. Both the CobT protein of S. typhimurium and its Pseudomonas homolog have been shown in vitro to catalyze the transfer of ribose phosphate from nicotinate mononucleotide to DMB. This reaction does not contribute to DMB synthesis but rather is the first step in joining DMB to the corrin ring compound cobinamide. Thus, the phenotype of Salmonella cobT mutants conflicts with the reported activity of the affected enzyme, while Pseudomonas mutants have the expected phenotype. J. R. Trzebiatowski, G. A. O'Toole, and J. C. Escalante Semerena have suggested (J. Bacteriol. 176:3568-3575, 1994) that S. typhimurium possesses a second phosphoribosyltransferase activity (CobB) that requires a high concentration of DMB for its activity. We support that suggestion and, in addition, provide evidence that the CobT protein catalyzes both the synthesis of DMB and transfer of ribose phosphate. Some cobT mutants appear defective only in DMB synthesis, since they grow on low levels of DMB and retain their CobII phenotype in the presence of a cobB mutation. Other mutants including those with deletions, appear defective in transferase, since they require a high level of DMB (to activate CobB) and, in combination with a cobB mutation, they eliminate the ability to join DMB and cobinamide. Immediately downstream of the cob operon is a gene (called ORF in this study) of unknown function whose mutants have no detected phenotype. Just counterclockwise of ORF is an asparagine tRNA gene (probably asnU). Farther counterclockwise, a serine tRNA gene (serU or supD) is weakly cotransducible with the cobT gene.     

The cobT gene of Salmonella typhimurium encodes the NaMN: 5,6-dimethylbenzimidazole phosphoribosyltransferase responsible for the synthesis of N1-(5-phospho-alpha-D-ribosyl)-5,6-dimethylbenzimidazole, an intermediate in the synthesis of the nucleotide loop of cobalamin.

We present in vitro evidence which demonstrates that CobT is the nicotinate nucleotide:5,6-dimethylbenzimidazole (DMB) phosphoribosyltransferase (EC 2.4.2.21) that catalyzes the synthesis of N1-(5-phospho-alpha-D-ribosyl)-5,6-dimethylbenzimidazole, a biosynthetic intermediate of the pathway that assembles the nucleotide loop of cobalamin in Salmonella typhimurium. Mutants previously isolated as DMB auxotrophs are shown by physical and genetic mapping studies and complementation studies to carry lesions in cobT. Explanations for this unexpected phenotype of cobT mutants are discussed. The expected nucleotide loop assembly phenotype of cobT mutants can be observed only in a specific genetic background, i.e., cobB deficient, an observation that is consistent with the existence of an alternative CobT function (G. A. O'Toole, M. R. Rondon, and J. C. Escalante-Semerena, J. Bacteriol. 175:3317-3326, 1993). Computer analysis of CobT homologs showed that at the amino acid level, enteric CobT proteins were 80% identical whereas Pseudomonas denitrificans and Rhizobium meliloti CobT proteins were 95% identical. Interestingly, the degree of identity between enteric and nonenteric CobT homologs was only 30%. The same pattern of homologies was reported for the S. typhimurium CobA, Escherichia coli BtuR, and P. denitrificans CobO proteins (S.-J. Suh and J.C. Escalante-Semerena, Gene 129:93-97, 1993), suggesting evolutionary divergence between the cob genes found in the enteric bacteria E. coli and S. typhimurium and those found in P. denitrificans and R. meliloti.     

A functional chimeric membrane subunit of an ion-translocating ATPase

A chimeric transport protein was made by expression of a fusion of thearsB genes fromEscherichia coli plasmid R773 andStaphylococcus aureus plasmid pI258. The two genes were fused to encode a functional protein with first eight membrane spanning -helices of theS. aureus and the last four helices of theE. coli protein. The hybrid protein provided arsenite resistance and transport. When anarsA gene was expressed in trans with the ArsB proteins encoded by the R773, pI258 and fusion genes, arsenite efflux was dependent on chemical but not electrochemical energy. The Ars system is hypothesized to be a novel transport system that functions as a primary ATP-driven pump or a secondary carrier, depending on the subunit composition of the complex.     

Hybridization in the Sida ovata complex (Malvaceae) III. Evidences from seed micro-morphology and seed protein analysis

In this paper, we present a detailed analysis of seed morphological characters and seed protein composition of two closely related species belonging to the Sida ovata complex (Malvaceae), Sida ovata Forssk. and S. tiagii Bhandari. Seed protein profiling was performed by sodium dodesyl sulphate–polyacrylamide gel electrophoresis and size exclusion‐fast protein liquid chromatography. Our data provide additional information to strengthen the existence of hybridization between Sida ovata Forssk. and S. tiagii Bhandari in Pakistan.     

Biochemical characterization of a novel ArsA ATPase complex from Alkaliphilus metalliredigens QYMF

The two putative ars operons in Alkaliphilus metalliredigens QYMF are distinctive in that the arsA gene is split in halves, amarsA1 and amarsA2, and, acr3 but not an arsB gene coexists with arsA. Heterologous expression of one of the A. metalliredigensars operons (ars1) conferred arsenite but not antimonite resistance to ΔarsEscherichia coli. Only the co-expressed AmArsA1 and AmArsA2 displayed arsenite or antimonite stimulated ATPase activity. The results show that AmArsA1-AmArsA2 interaction is needed to form the functional ArsA ATPase. This novel AmArsA1-AmArsA2 complex may provide insight in how it participates with Acr3 in arsenite detoxification.     

Effective Anomerisation of 2′‐Deoxyadenosine Derivatives During Disaccharide Nucleoside Synthesis

The formation of a disaccharide nucleoside (11) by O3′‐glycosylation of 5′‐O‐protected 2′‐deoxyadenosine or its N ⁶‐benzoylated derivative has been observed to be accompanied by anomerisation to the corresponding α‐anomeric product (12). The latter reaction can be explained by instability of the N‐glycosidic bond of purine 2′‐deoxynucleosides in the presence of Lewis acids. An independent study on the anomerisation of partly blocked 2′‐deoxyadenosine has been carried out. Additionally, transglycosylation has been utilized in the synthesis of 3′‐O‐β‐d‐ribofuranosyl‐2′‐deoxyadenosines and its α‐anomer.     

Investigation into FlhFG reveals distinct features of FlhF in regulating flagellum polarity in Shewanella oneidensis

Rod‐shaped bacterial cells are polarized, with many organelles confined to a polar cellular site. In polar flagellates, FlhF and FlhG, a multiple‐domain (B‐N‐G) GTPase and a MinD‐like ATPase respectively, function as a cognate pair to regulate flagellar localization and number as revealed in Vibrio and Pseudomonas species. In this study, we show that FlhFG of Shewanella oneidensis (SoFlhFG), a monotrichous γ‐proteobacterium renowned for respiratory diversity, also play an important role in the flagellar polar placement and number control. Despite this, SoFlhFG exhibit distinct features that are not observed in the characterized counterparts. Most strikingly, the G domain of SoFlhF determines the polar placement, contrasting the N domain of the Vibrio cholerae FlhF. The SoFlhF N domain in fact counteracts the function of the G domain with respect to the terminal targeting in the absence of the B domain. We further show that GTPase activity of SoFlhF is essential for motility but not positioning. Overall, our results suggest that mechanisms underlying the polar placement of organelles appear to be diverse, even for evolutionally relatively conserved flagellum.     

Remodulation of central carbon metabolic pathway in response to arsenite exposure in Rhodococcus sp. strain NAU‐1

Arsenite‐tolerant bacteria were isolated from an organic farm of Navsari Agricultural University (NAU), Gujarat, India (Latitude: 20°55′39.04″N; Longitude: 72°54′6.34″E). One of the isolates, NAU‐1 (aerobic, Gram‐positive, non‐motile, coccobacilli), was hyper‐tolerant to arsenite (As^III, 23 mM) and arsenate (As^V, 180 mM). 16S rRNA gene of NAU‐1 was 99% similar to the 16S rRNA genes of Rhodococcus (Accession No. HQ659188). Assays confirmed the presence of membrane bound arsenite oxidase and cytoplasmic arsenate reductase in NAU‐1. Genes for arsenite transporters (arsB and ACR3(1)) and arsenite oxidase gene (aoxB) were confirmed by PCR. Arsenite oxidation and arsenite efflux genes help the bacteria to tolerate arsenite. Specific activities of antioxidant enzymes (catalase, ascorbate peroxidase, superoxide dismutase and glutathione S‐transferase) increased in dose‐dependent manner with arsenite, whereas glutathione reductase activity decreased with increase in As^III concentration. Metabolic studies revealed that Rhodococcus NAU‐1 produces excess of gluconic and succinic acids, and also activities of glucose dehydrogenase, phosphoenol pyruvate carboxylase and isocitrate lyase were increased, to cope with the inhibited activities of glucose‐6‐phosphate dehydrogenase, pyruvate dehydrogenase and α‐ketoglutarate dehydrogenase enzymes respectively, in the presence of As^III. Enzyme assays revealed the increase in direct oxidative and glyoxylate pathway in Rhodococcus NAU‐1 in the presence of As^III.     

Development and laboratory evaluation of chemically‐based baited ovitrap for the monitoring of Aedes aegypti

Aedes (Stegomyia) aegypti is considered to be the most important dengue vector worldwide. Studies were conducted to design and evaluate a chemically‐based baited ovitrap for monitoring Ae. aegypti under laboratory conditions. Several known chemical attractants and three types of ovitraps (ovitraps A, B, and C) were evaluated throughout the oviposition bioassays. Oviposition responses of gravid female Ae. aegypti were evaluated to n‐heneicosane, 3‐methylindole (skatole), 4‐methylphenol (p‐cresol), and phenol. Female Ae. aegypti were attracted to all the evaluated compounds. Among them, n‐heneicosane at a concentration of 10 ppm (mg/l), skatole from 50 to 1000 ppm, p‐cresol at 100 ppm, and phenol at 50 ppm showed a significant positive oviposition response. A blend of the four chemical attractants increased the oviposition response; 67% of the eggs were deposited in the treatment compared to the control. Female Ae. aegypti were signi?cantly more attracted to ovitrap A loaded with the four‐component synthetic blend compared to the standard ovitrap in the oviposition bioassays. The compound used in ovitrap A retained its attractant property for up to three days. The chemically‐based baited ovitrap may be considered as an option to be integrated during the monitoring of dengue virus vectors in México.