scholarly journals Selective Removal of Aberrant Extender Units by a Type II Thioesterase for Efficient FR-008/Candicidin Biosynthesis in Streptomyces sp. Strain FR-008

2008 ◽  
Vol 74 (23) ◽  
pp. 7235-7242 ◽  
Author(s):  
Yongjun Zhou ◽  
Qingqing Meng ◽  
Delin You ◽  
Jialiang Li ◽  
Shi Chen ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
Biochemical Analysis ◽  
Wild Type Strain ◽  
Type I ◽  
Type Ii ◽  
Wild Type ◽  
Acyl Carrier Proteins ◽  
Peptide Synthetase ◽  
In The Wild

ABSTRACT Gene fscTE, encoding a putative type II thioesterase (TEII), was associated with the FR-008/candicidin gene cluster. Deletion of fscTE reduced approximately 90% of the FR-008/candicidin production, while the production level was well restored when fscTE was added back to the mutant in trans. FscTE was unable to compensate for the release of the maturely elongated polyketide as site-directed inactivation of the type I thioesterase (TEI) totally abolished FR-008/candicidin production. Direct biochemical analysis of FscTE in parallel with its homologue TylO from the tylosin biosynthetic pathway demonstrated their remarkable preferences for acyl-thioesters (i.e., propionyl-S-N-acetylcysteamine [SNAC] over methylmalonyl-SNAC and acetyl-SNAC over malonyl-SNAC) and thus concluded that TEII could maintain effective polyketide biosynthesis by selectively removing the nonelongatable residues bound to acyl carrier proteins. Overexpression of FscTE under the strong constitutive ermE*p promoter in the wild-type strain did not suppress FR-008/candicidin formation, which confirmed its substrate specificity in vivo. Furthermore, successful complementation of the fscTE mutant was obtained with fscTE and tylO, whereas no complementation was detected with nonribosomal peptide synthetase (NRPS) TEII tycF and srfAD, reflecting substrate specificities of TEIIs distinctive from those of either polyketide synthases or NRPSs.

scholarly journals Involvement of H-NS in Transpositional Recombination Mediated by IS1

2001 ◽  
Vol 183 (8) ◽  
pp. 2476-2484 ◽  
Author(s):  
Yasuyuki Shiga ◽  
Yasuhiko Sekine ◽  
Yasunobu Kano ◽  
Eiichi Ohtsubo
Keyword(s):  
Dna Binding ◽  
Wild Type Strain ◽  
Integration Host Factor ◽  
Deficient Mutant ◽  
Wild Type ◽  
Transposase Gene ◽  
In The Wild ◽  
Transposition Reaction ◽  
Target Plasmid

ABSTRACT IS1, the smallest active transposable element in bacteria, encodes a transposase that promotes inter- and intramolecular transposition. Host-encoded factors, e.g., histone-like proteins HU and integration host factor (IHF), are involved in the transposition reactions of some bacterial transposable elements. Host factors involved in the IS1 transposition reaction, however, are not known. We show that a plasmid with an IS1 derivative that efficiently produces transposase did not generate miniplasmids, the products of intramolecular transposition, in mutants deficient in a nucleoid-associated DNA-binding protein, H-NS, but did generate them in mutants deficient in histone-like proteins HU, IHF, Fis, and StpA. Nor did IS1 transpose intermolecularly to the target plasmid in the H-NS-deficient mutant. The hns mutation did not affect transcription from the indigenous promoter of IS1 for the expression of the transposase gene. These findings show that transpositional recombination mediated by IS1 requires H-NS but does not require the HU, IHF, Fis, or StpA protein in vivo. Gel retardation assays of restriction fragments of IS1-carrying plasmid DNA showed that no sites were bound preferentially by H-NS within the IS1 sequence. The central domain of H-NS, which is involved in dimerization and/or oligomerization of the H-NS protein, was important for the intramolecular transposition of IS1, but the N- and C-terminal domains, which are involved in the repression of certain genes and DNA binding, respectively, were not. The SOS response induced by the IS1 transposase was absent in the H-NS-deficient mutant strain but was present in the wild-type strain. We discuss the possibility that H-NS promotes the formation of an active IS1 DNA-transposase complex in which the IS1 ends are cleaved to initiate transpositional recombination through interaction with IS1 transposase.

scholarly journals Involvement of the XpsN Protein in Formation of the XpsL-XpsM Complex in Xanthomonas campestris pv. campestris Type II Secretion Apparatus

2001 ◽  
Vol 183 (2) ◽  
pp. 528-535 ◽  
Author(s):  
Hsien-Ming Lee ◽  
Shiaw-Wei Tyan ◽  
Wei-Ming Leu ◽  
Ling-Yun Chen ◽  
David Chanhen Chen ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Mutant Strain ◽  
Type Strain ◽  
Xanthomonas Campestris ◽  
Wild Type Strain ◽  
Type Ii ◽  
Wild Type ◽  
In The Wild ◽  
Steady State Levels ◽  
Type Gene

ABSTRACT The xps gene cluster is required for the second step of type II protein secretion in Xanthomonas campestrispv. campestris. Deletion of the entire gene cluster caused accumulation of secreted proteins in the periplasm. By analyzing protein abundance in the chromosomal mutant strains, we observed mutual dependence for normal steady-state levels between the XpsL and the XpsM proteins. The XpsL protein was undetectable in total lysate prepared from thexpsM mutant strain, and vice versa. Introduction of the wild-type xpsM gene carried on a plasmid into thexpsM mutant strain was sufficient for reappearance of the XpsL protein, and vice versa. Moreover, both XpsL and XpsM proteins were undetectable in the xpsN mutant strain. They were recovered either by reintroducing the wild-type xpsNgene or by introducing extra copies of wild-type xpsL orxpsM individually. Overproduction of wild-type XpsL and -M proteins simultaneously, but not separately, in the wild-type strain of X. campestris pv. campestris caused inhibition of secretion. Complementation of an xpsL orxpsM mutant strain with a plasmid-borne wild-type gene was inhibited by coexpression of XpsL and XpsM. The presence of the xpsN gene on the plasmid along with thexpsL and the xpsM genes caused more severe inhibition in both cases. Furthermore, complementation of thexpsN mutant strain was also inhibited. In both the wild-type strain and a strain with the xps gene cluster deleted (XC17433), carrying pCPP-LMN, which encodes all three proteins, each protein coprecipitated with the other two upon immunoprecipitation. Expression of pairwise combinations of the three proteins in XC17433 revealed that the XpsL-XpsM and XpsM-XpsN pairs still coprecipitated, whereas the XpsL-XpsN pair no longer coprecipitated.

scholarly journals Regulation of ppk Expression and In Vivo Function of Ppk in Streptomyces lividans TK24

2006 ◽  
Vol 188 (17) ◽  
pp. 6269-6276 ◽  
Author(s):  
Sofiane Ghorbel ◽  
Aleksey Smirnov ◽  
Hichem Chouayekh ◽  
Brice Sperandio ◽  
Catherine Esnault ◽  
...  
Keyword(s):  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Sufficient Conditions ◽  
Streptomyces Lividans ◽  
Antibiotic Biosynthesis ◽  
Wild Type ◽  
In The Wild

ABSTRACT The ppk gene of Streptomyces lividans encodes an enzyme catalyzing, in vitro, the reversible polymerization of the γ phosphate of ATP into polyphosphate and was previously shown to play a negative role in the control of antibiotic biosynthesis (H. Chouayekh and M. J. Virolle, Mol. Microbiol. 43:919-930, 2002). In the present work, some regulatory features of the expression of ppk were established and the polyphosphate content of S. lividans TK24 and the ppk mutant was determined. In Pi sufficiency, the expression of ppk was shown to be low but detectable. DNA gel shift experiments suggested that ppk expression might be controlled by a repressor using ATP as a corepressor. Under these conditions, short acid-soluble polyphosphates accumulated upon entry into the stationary phase in the wild-type strain but not in the ppk mutant strain. The expression of ppk under Pi-limiting conditions was shown to be much higher than that under Pi-sufficient conditions and was under positive control of the two-component system PhoR/PhoP. Under these conditions, the polyphosphate content of the cell was low and polyphosphates were reproducibly found to be longer and more abundant in the ppk mutant strain than in the wild-type strain, suggesting that Ppk might act as a nucleoside diphosphate kinase. In light of our results, a novel view of the role of this enzyme in the regulation of antibiotic biosynthesis in S. lividans TK24 is proposed.

scholarly journals relA Is Required for Actinomycin Production in Streptomyces antibioticus

1999 ◽  
Vol 181 (12) ◽  
pp. 3824-3829 ◽  
Author(s):  
Shannan Hoyt ◽  
George H. Jones
Keyword(s):  
Biosynthetic Pathway ◽  
Wild Type Strain ◽  
Streptomyces Coelicolor ◽  
Production Medium ◽  
Wild Type ◽  
Streptomyces Antibioticus ◽  
Phenoxazinone Synthase ◽  
Key Enzyme ◽  
In The Wild ◽  
Phosphate Medium

ABSTRACT The relA gene from Streptomyces antibioticus has been cloned and sequenced. The gene encodes a protein with an M r of 93,653, which is 91% identical to the corresponding protein from Streptomyces coelicolor. Disruption of S. antibioticus relAproduces a strain which grows significantly more slowly on actinomycin production medium than the wild type or a disruptant to which the intact relA gene was restored. Moreover, the disruptant was unable to accumulate ppGpp to the levels observed during the normal course of growth and actinomycin production in the wild type. The strain containing the disrupted relA gene did not produce actinomycin and contained significantly lower levels of the enzyme phenoxazinone synthase than the wild-type strain. Actinomycin synthetase I, a key enzyme in the actinomycin biosynthetic pathway, was undetectable in the relA disruptant. Growth of the disruptant on low-phosphate medium did not restore actinomycin production.

scholarly journals Involvement of the SppA1 Peptidase in Acclimation to Saturating Light Intensities in Synechocystis sp. Strain PCC 6803

2004 ◽  
Vol 186 (12) ◽  
pp. 3991-3999 ◽  
Author(s):  
E. Pojidaeva ◽  
V. Zinchenko ◽  
S. V. Shestakov ◽  
A. Sokolenko
Keyword(s):  
Light Intensity ◽  
Wild Type Strain ◽  
Wild Type ◽  
Additional Mechanism ◽  
Gene Encoding ◽  
Light Intensities ◽  
In The Wild ◽  
Pcc 6803

ABSTRACT The sll1703 gene, encoding an Arabidopsis homologue of the thylakoid membrane-associated SppA peptidase, was inactivated by interposon mutagenesis in Synechocystis sp. strain PCC 6803. Upon acclimation from a light intensity of 50 to 150 μE m−2 s−1, the mutant preserved most of its phycobilisome content, whereas the wild-type strain developed a bleaching phenotype due to the loss of about 40% of its phycobiliproteins. Using in vivo and in vitro experiments, we demonstrate that the ΔsppA1 strain does not undergo the cleavage of the LR 33 and LCM 99 linker proteins that develops in the wild type exposed to increasing light intensities. We conclude that a major contribution to light acclimation under a moderate light regime in cyanobacteria originates from an SppA1-mediated cleavage of phycobilisome linker proteins. Together with changes in gene expression of the major phycobiliproteins, it contributes an additional mechanism aimed at reducing the content in phycobilisome antennae upon acclimation to a higher light intensity.

scholarly journals Interferons Regulate the Phenotype of  Wild-type and Mutant Herpes Simplex Viruses In Vivo

1999 ◽  
Vol 189 (4) ◽  
pp. 663-672 ◽  
Author(s):  
David A. Leib ◽  
Travis E. Harrison ◽  
Kathleen M. Laslo ◽  
Michael A. Machalek ◽  
Nathaniel J. Moorman ◽  
...  
Keyword(s):  
Cell Culture ◽  
Herpes Simplex ◽  
Host Factors ◽  
Productive Infection ◽  
Cell Protein ◽  
Trigeminal Ganglia ◽  
Type I ◽  
Type Ii ◽  
Wild Type

Mechanisms responsible for neuroattenuation of herpes simplex virus (HSV) have been defined previously by studies of mutant viruses in cultured cells. The hypothesis that null mutations in host genes can override the attenuated phenotype of null mutations in certain viral genes was tested. Mutants such as those in infected cell protein (ICP) 0, thymidine kinase, ribonucleotide reductase, virion host shutoff, and ICP34.5 are reduced in their capacity to replicate in nondividing cells in culture and in vivo. The replication of these viruses was examined in eyes and trigeminal ganglia for 1–7 d after corneal inoculation in mice with null mutations (−/−) in interferon receptors (IFNR) for type I IFNs (IFN-α/βR), type II IFN (IFN-γR), and both type I and type II IFNs (IFN-α/β/γR). Viral titers in eyes and ganglia of IFN-γR−/− mice were not significantly different from congenic controls. However, in IFN-α/βR−/− or IFN-α/β/γR−/− mice, growth of all mutants, including those with significantly impaired growth in cell culture, was enhanced by up to 1,000-fold in eyes and trigeminal ganglia. Blepharitis and clinical signs of infection were evident in IFN-α/βR−/− and IFN-α/β/γR−/− but not control mice for all viruses. Also, IFNs were shown to significantly reduce productive infection of, and spread from intact, but not scarified, corneas. Particularly striking was restoration of near-normal trigeminal ganglion replication and neurovirulence of an ICP34.5 mutant in IFN-α/βR−/− mice. These data show that IFNs play a major role in limiting mutant and wild-type HSV replication in the cornea and in the nervous system. In addition, the in vivo target of ICP34.5 may be host IFN responses. These experiments demonstrate an unsuspected role for host factors in defining the phenotypes of some HSV mutants in vivo. The phenotypes of mutant viruses therefore cannot be interpreted based solely upon studies in cell culture but must be considered carefully in the context of host factors that may define the in vivo phenotype.

scholarly journals Acinetobacter baumannii Is Dependent on the Type II Secretion System and Its Substrate LipA for Lipid Utilization andIn VivoFitness

2015 ◽  
Vol 198 (4) ◽  
pp. 711-719 ◽  
Author(s):  
Tanya L. Johnson ◽  
Ursula Waack ◽  
Sara Smith ◽  
Harry Mobley ◽  
Maria Sandkvist
Keyword(s):  
Acinetobacter Baumannii ◽  
Wild Type Strain ◽  
Type Ii Secretion ◽  
Long Chain Fatty Acids ◽  
Type Ii ◽  
Wild Type ◽  
Extracellular Lipase ◽  
Content Type ◽  
Long Chain

ABSTRACTGram-negative bacteria express a number of sophisticated secretion systems to transport virulence factors across the cell envelope, including the type II secretion (T2S) system. Genes for the T2S components GspC through GspN and PilD are conserved among isolates ofAcinetobacter baumannii, an increasingly common nosocomial pathogen that is developing multidrug resistance at an alarming rate. In contrast to most species, however, the T2S genes are dispersed throughout the genome rather than linked into one or two operons. Despite this unique genetic organization, we show here that theA. baumanniiT2S system is functional. Deletion ofgspDorgspEinA. baumanniiATCC 17978 results in loss of secretion of LipA, a lipase that breaks down long-chain fatty acids. Due to a lack of extracellular lipase, thegspDmutant, thegspEmutant, and alipAdeletion strain are incapable of growth on long-chain fatty acids as a sole source of carbon, while their growth characteristics are indistinguishable from those of the wild-type strain in nutrient-rich broth. Genetic inactivation of the T2S system and its substrate, LipA, also has a negative impact onin vivofitness in a neutropenic murine model for bacteremia. Both thegspDandlipAmutants are outcompeted by the wild-type strain as judged by their reduced numbers in spleen and liver following intravenous coinoculation. Collectively, our findings suggest that the T2S system plays a hitherto-unrecognized role inin vivosurvival ofA. baumanniiby transporting a lipase that may contribute to fatty acid metabolism.IMPORTANCEInfections by multidrug-resistantAcinetobacter baumanniiare a growing health concern worldwide, underscoring the need for a better understanding of the molecular mechanisms by which this pathogen causes disease. In this study, we demonstrated thatA. baumanniiexpresses a functional type II secretion (T2S) system that is responsible for secretion of LipA, an extracellular lipase required for utilization of exogenously added lipids. The T2S system and the secreted lipase supportin vivocolonization and thus contribute to the pathogenic potential ofA. baumannii.

scholarly journals The Hydroxamate Siderophore Rhequichelin Is Required for Virulence of the Pathogenic Actinomycete Rhodococcus equi

2012 ◽  
Vol 80 (12) ◽  
pp. 4106-4114 ◽  
Author(s):  
Raúl Miranda-CasoLuengo ◽  
Garry B. Coulson ◽  
Aleksandra Miranda-CasoLuengo ◽  
José A. Vázquez-Boland ◽  
Mary K. Hondalus ◽  
...  
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Iron Acquisition ◽  
Iron Chelator ◽  
Rhodococcus Equi ◽  
Wild Type ◽  
Content Type ◽  
Peptide Synthetase ◽  
Tailoring Enzymes

ABSTRACTWe previously showed that the facultative intracellular pathogenRhodococcus equiproduces a nondiffusible and catecholate-containing siderophore (rhequibactin) involved in iron acquisition during saprophytic growth. Here, we provide evidence that therhbABCDEcluster directs the biosynthesis of a hydroxamate siderophore, rhequichelin, that plays a key role in virulence. TherhbCgene encodes a nonribosomal peptide synthetase that is predicted to produce a tetrapeptide consisting ofN5-formyl-N5-hydroxyornithine, serine,N5-hydroxyornithine, andN5-acyl-N5-hydroxyornithine. The otherrhbgenes encode putative tailoring enzymes mediating modification of ornithine residues incorporated into the hydroxamate product of RhbC. Transcription ofrhbCwas upregulated during growth in iron-depleted medium, suggesting that it plays a role in iron acquisition. This was confirmed by deletion ofrhbCD, rendering the resulting strainR. equiSID2 unable to grow in the presence of the iron chelator 2,2-dipyridyl. Supernatant of the wild-type strain rescued the phenotype ofR. equiSID2. The importance of rhequichelin in virulence was highlighted by the rapid increase in transcription levels ofrhbCfollowing infection and the inability ofR. equiSID2 to grow within macrophages. Unlike the wild-type strain,R. equiSID2 was unable to replicatein vivoand was rapidly cleared from the lungs of infected mice. Rhequichelin is thus a key virulence-associated factor, although nonpathogenicRhodococcusspecies also appear to produce rhequichelin or a structurally closely related compound. Rhequichelin biosynthesis may therefore be considered an example of cooption of a core actinobacterial trait in the evolution ofR. equivirulence.

Evidence of cyclic GMP may regulate the association of myosin II heavy chain with the cytoskeleton by inhibiting its phosphorylation

1991 ◽  
Vol 98 (4) ◽  
pp. 483-490
Author(s):  
G. Liu ◽  
P.C. Newell
Keyword(s):  
Cyclic Amp ◽  
Heavy Chain ◽  
Cyclic Gmp ◽  
Parental Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Myosin Ii ◽  
Wild Type ◽  
In The Wild

Previous studies have implicated cyclic GMP in the regulation of myosin II heavy chain (MHC) association with the cytoskeleton in Dictyostelium discoideum. Here we provide evidence that cyclic GMP may regulate MHC association with the cytoskeleton through MHC phosphorylation. Comparative data are presented of MHC phosphorylation in the wild-type strain NC4, the parental strain XP55 and streamer mutants NP368 and NP377. Using an anti-MHC monoclonal antibody to immunoprecipitate MHC from [32P]phosphate-labelled developing cells, we found that cyclic AMP stimulation of the wild-type strain NC4 and parental strain XP55 induced MHC phosphorylation in vivo. A peak of phosphorylation was observed at 30–40 s, followed by a gradual decrease to basal level at 160 s. In contrast, in both of the streamer mutants NP368 and NP377 (which have prolonged cyclic GMP accumulation and prolonged MHC association with the cytoskeleton), the phosphorylation of MHC was delayed and did not form a peak until 60–80 s after cyclic AMP stimulation. We also found that cytoskeletal MHC showed only minor phosphorylation, the majority of the phosphorylated MHC being found in the cytosol. We present a model to account for these results in which cyclic GMP regulates MHC association with the cytoskeleton by regulating the phosphorylation/dephosphorylation cycle of MHC in these cells.

scholarly journals Target Preference of 15 Quinolones against Staphylococcus aureus, Based on Antibacterial Activities and Target Inhibition

2001 ◽  
Vol 45 (12) ◽  
pp. 3544-3547 ◽  
Author(s):  
Masaya Takei ◽  
Hideyuki Fukuda ◽  
Ryuta Kishii ◽  
Masaki Hosaka
Keyword(s):  
Staphylococcus Aureus ◽  
Type Strain ◽  
Wild Type Strain ◽  
Dna Gyrase ◽  
Antibacterial Activities ◽  
Type I ◽  
Type Ii ◽  
Wild Type ◽  
Topoisomerase Iv ◽  
Mutant Strains

ABSTRACT The antibacterial activities and target inhibition of 15 quinolones against grlA and gyrA mutant strains were studied. The strains were obtained from wild-type Staphylococcus aureus MS5935 by selection with norfloxacin and nadifloxacin, respectively. The antibacterial activities of most quinolones against both mutant strains were lower than those against the wild-type strain. The ratios of MICs for the gyrA mutant strain to those for the grlA mutant strain (MIC ratio) varied from 0.125 to 4. The ratios of 50% inhibitory concentrations (IC50s) of quinolones against topoisomerase IV to those against DNA gyrase (IC50 ratios) also varied, from 0.177 to 5.52. A significant correlation between the MIC ratios and the IC50ratios was observed (r = 0.919; P < 0.001). These results suggest that the antibacterial activities of quinolones against the wild-type strain are involved not only in topoisomerase IV inhibition but also in DNA gyrase inhibition and that the target preference in the wild-type strain can be anticipated by the MIC ratios. Based on the MIC ratios, the quinolones were classified into three categories. Type I quinolones (norfloxacin, enoxacin, fleroxacin, ciprofloxacin, lomefloxacin, trovafloxacin, grepafloxacin, ofloxacin, and levofloxacin) had MIC ratios of <1, type II quinolones (sparfloxacin and nadifloxacin) had MIC ratios of >1, and type III quinolones (gatifloxacin, pazufloxacin, moxifloxacin, and clinafloxacin) had MIC ratios of 1. Type I and type II quinolones seem to prefer topoisomerase IV and DNA gyrase, respectively. Type III quinolones seem to target both enzymes at nearly the same level in bacterial cells (a phenomenon known as the dual-targeting property), and their IC50 ratios were approximately 2.

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