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Archives of Microbiology (v.183, #4)
The Bacillus subtilis desaturase: a model to understand phospholipid modification and temperature sensing by María C. Mansilla; Diego de Mendoza (pp. 229-235).
Most fatty acid desaturases are members of a large superfamily of integral membrane, O2-dependent, iron-containing enzymes that insert double bonds into previously synthesized fatty acyl chains. The cold shock-induced, membrane-bound desaturase from Bacillus subtilis (Δ5-Des) uses existing phospholipids as substrates to introduce a cis-double bond at the fifth position of the fatty acyl chain. While essentially no three-dimensional structural information is available for these difficult-to-purify enzymes, experimental analysis of the topology of Δ5-Des has provided a model that might be extended to most acyl-lipid desaturases. In addition, studies of the cold-induced expression of Δ5-Des led to the identification of a two-component system composed of a membrane-associated kinase, DesK, and a transcriptional regulator, DesR, which stringently controls the transcription of the des gene, coding for the desaturase. A model for sensing and transduction of low-temperature signals has emerged from our results, which we discuss in the context of transcriptional regulation of membrane lipid fluidity homeostasis.
Keywords: Regulation of membrane lipid synthesis; Desaturase topology; Signal transduction
The ability of acidic pH, growth inhibitors, and glucose to increase the proton motive force and energy spilling of amino acid-fermenting Clostridium sporogenes MD1 cultures by Michael D. Flythe; James B. Russell (pp. 236-242).
Clostridium sporogenes MD1 grew rapidly with peptides and amino acids as an energy source at pH 6.7. However, the proton motive force (Δp) was only −25 mV, and protonophores did not inhibit growth. When extracellular pH was decreased with HCl, the chemical gradient of protons (ZΔpH) and the electrical membrane potential (ΔΨ) increased. The Δp was −125 mV at pH 4.7, even though growth was not observed. At pH 6.7, glucose addition did not cause an increase in growth rate, but ΔΨ increased to −70 mV. Protein synthesis inhibitors also significantly increased ΔΨ. Non-growing, arginine-energized cells had a ΔΨ of −80 mV at pH 6.7 or pH 4.7, but ΔΨ was not detected if the F1F0 ATPase was inhibited. Arginine-energized cells initiated growth if other amino acids were added at pH 6.7, and ΔΨ and ATP declined. At pH 4.7, ATP production remained high. However, growth could not be initiated, and neither ΔΨ nor the intracellular ATP concentration declined. Based on these results, it appears that C. sporogenes MD1 does not need a large Δp to grow, and Δp appears to serve as a mechanism of ATP dissipation or energy spilling.
Keywords: Clostridium sporogenesClostridium botulinumProton motive force; Intracellular pH; Membrane potential
Demonstration of interactions among Myxococcus xanthus Dif chemotaxis-like proteins by the yeast two-hybrid system by Zhaomin Yang; Zhuo Li (pp. 243-252).
The Myxococcus xanthus dif locus encodes several bacterial chemotaxis homologues that are crucial for fibril exopolysaccharide (EPS) production, social gliding motility, and fruiting body development. In primary sequence, DifA is homologous to methyl-accepting chemotaxis protein, DifC to CheW, DifD to CheY, DifE to CheA, and DifG to CheC. In this study, the interactions among the Dif chemotaxis-like proteins were investigated using the yeast two-hybrid (Y2H) system. DifC was found to interact with both DifA and DifE. Using a modified Y2H or a “three-hybrid” system, it was demonstrated that DifC is capable of mediating the formation of DifA, DifC, and DifE ternary protein complexes. The conserved domains of DifE, based on sequence analysis, likely reflect functional conservations of CheA-type kinases, because its P2 domain interacts with DifD, P5 with DifC, and the P3 domain appears to dimerize. Similarly, C-terminal regions of DifA appear to dimerize as well. In addition, DifG was found to interact with DifD, which is consistent with the hypothesis that DifG is a phosphatase of DifD-phosphate. These findings support the models in which Dif proteins constitute a unique chemotaxis-like signal transduction pathway with central functions in regulating EPS production in M. xanthus.
Keywords: Myxococcus xanthusFibril exopolysaccharide; Dif; Chemotaxis; Yeast two-hybrid; Protein interaction
Two of the three groEL homologues in Rhizobium leguminosarum are dispensable for normal growth by F. Rodríguez-Quiñones; M. Maguire; E. J. Wallington; Phillip S. Gould; V. Yerko; J. A. Downie; P. A. Lund (pp. 253-265).
Although many bacteria contain only a single groE operon encoding the essential chaperones GroES and GroEL, examples of bacteria containing more than one groE operon are common. The root-nodulating bacterium Rhizobium leguminosarum contains at least three operons encoding homologues to Escherichia coli GroEL, referred to as Cpn60.1, Cpn60.2 and Cpn60.3, respectively. We report here a detailed analysis of the requirement for and relative levels of these three proteins. Cpn60.1 is present at higher levels than Cpn60.2, and Cpn60.3 protein could not be detected under any conditions although the cpn60.3 gene is transcribed under anaerobic conditions. Insertion mutations could not be constructed in cpn60.1 unless a complementing copy was present, showing that this gene is essential for growth under the conditions used here. Both cpn60.2 and cpn60.3 could be inactivated with no loss of viability, and a double cpn60.2 cpn60.3 mutant was also constructed which was fully viable. Thus only Cpn60.1 is required for growth of this organism.
Keywords: Rhizobium leguminosarumGroEL; Chaperonins; Heat shock
Substituent effects on the oxidation of substituted biphenyl congeners by type II methanotroph strain CSC1 by A. S. Lindner; J. D. Semrau; P. Adriaens (pp. 266-276).
The oxidation potential of type II groundwater methanotroph, strain CSC1, expressing soluble methane monooxygenase, was measured in the presence of 10 ortho-substituted biphenyls with varying electronics, sterics, and hydrophobicity character for comparison with type II Methylosinus trichosporium OB3b. Strain CSC1 showed faster rates with all compounds tested, with the exception of 2-nitrobiphenyl, 2-hydroxybiphenyl, and 2-aminobiphenyl. Products of oxidation observed upon incubation of strain CSC1 with biphenyl and 2-hydroxybiphenyl were hydroxylated biphenyls that revealed less preference for the para position and different dihydroxylation positions, respectively, in comparison to those observed with M. trichosporium OB3b. Only the intramolecular hydrogen migration, or NIH-shift, product was observed in the case of 2-chlorobiphenyl, whereas M. trichosporium OB3b yielded a variety of chlorohydroxybiphenyls. Quantitative structure–biodegradation relationships constructed with the maximum observed oxygen uptake rates as a dependent variable and a variety of descriptors showed an influence of substituent electronic character on the oxidation activity of strain CSC1. However, compound hydrophobicity and not compound size, as was observed with M. trichosporium OB3b, was shown to influence rates to a greater extent. This suggests that transport of the compound through the cell membrane and to the sMMO active site is rate-determining for strain CSC1.
Keywords: Biodegradation; Methanotrophic oxidation; Molecular orbital descriptors; Multivariate analysis; Quantitative structure–biodegradation relationships; Soluble methane monooxygenase
Overexpression, purification and characterization of SimL, an amide synthetase involved in simocyclinone biosynthesis by Thomas Luft; Shu-Ming Li; Holger Scheible; Bernd Kammerer; Lutz Heide (pp. 277-285).
Simocyclinone D8 is a potent inhibitor of bacterial gyrase, produced by Streptomyces antibioticus Tü 6040. It contains an aminocoumarin moiety, similar to that of novobiocin, which is linked by an amide bond to a structurally complex acyl moiety, consisting of an aromatic angucycline polyketide nucleus, the deoxysugar olivose and a tetraene dicarboxylic acid. We have now investigated the enzyme SimL, responsible for the formation of the amide bond of simocyclinone. The gene was cloned, expressed in S. lividans T7, and the protein was purified to near homogeneity, and characterized. The 60 kDa protein catalyzed both the ATP-dependent activation of the acyl component as well as its transfer to the amino group of the aminocoumarin ring, with no requirement for a 4′-phosphopantetheinyl cofactor. Besides its natural substrate, simocyclinone C4, SimL also accepted a range of cinnamic and benzoic acid derivatives and several other, structurally very diverse acids. These findings make SimL a possible tool for the creation of new aminocoumarin antibiotics.
Keywords: Simocyclinone; Antibiotic biosynthesis; Amide synthetase
Staphylococcus aureus ClpYQ plays a minor role in stress survival by Dorte Frees; Line E. Thomsen; Hanne Ingmer (pp. 286-291).
Although bacteria lack the proteasome–ubiquitin proteolytic pathway, the homologue of the β-type proteasome subunit, ClpQ, is highly conserved among bacterial species. ClpQ associates with its ATPase partner, ClpY, to form a two-component protease, which also structurally resembles the 26S proteasome. Here we have disrupted clpQ and clpY of the versatile pathogen Staphylococcus aureus in order to examine the significance of the ClpYQ protease for growth under stress conditions. We found that the mutant, in contrast to the wild type, was unable to form colonies at very high temperatures. To our knowledge, this is the first-described phenotype of ClpYQ in Gram-positive bacteria. However, in the presence of puromycin and under all other stress conditions, tested growth of the clpYQ mutant cells was similar to growth of the wild type. Additionally, the absence of ClpYQ did not affect virulence as measured by a murine skin abscess model. Transcriptional analysis revealed that clpQ and clpY are expressed as part of a four-cistronic operon encompassing xerC and codY, and that expression is modestly induced by heat. In conclusion, our data indicates that ClpYQ plays only a secondary role in the degradation of non-native proteins in S. aureus.
Keywords: Proteolysis; HslU; HslV
Cytoplasmic contractions in growing fungal hyphae and their morphogenetic consequences by Cristina G. Reynaga-Peña; Salomón Bartnicki-García (pp. 292-300).
Video-enhanced light microscopy of the apical and subapical regions of growing hyphae of several fungal species revealed the existence of momentary synchronized motions of subcellular organelles. First discovered in a temperature-sensitive morphological mutant (ramosa-1) of Aspergillus niger, these seemingly spontaneous cytoplasmic contractions were also detected in wild-type hyphae of A. niger, Neurospora crassa, and Trichoderma atroviride. Cytoplasmic contractions in all fungi lasted about 1 s. Although the cytoplasm recovered its motility and appearance, the contraction usually led to drastic changes in Spitzenkörper (apical body) behavior and hyphal morphology, often both. Within 10 s after the contraction, the Spitzenkörper commonly became dislodged from its polar position; sometimes it disassembled into phase-dark and phase-light components; more commonly, it disappeared completely. Whether partial or complete, the dislocation of the Spitzenkörper was always accompanied by a sharp reduction or cessation of growth, and was usually followed by marked morphological changes that included bulbous hyphal tips, bulges in the hyphal profile, and formation of subapical and apical branches. The cytoplasmic contractions are vivid evidence that the most conspicuous cell organelles (membrane-bound) in living hyphae are interconnected via a contractile cytoskeletal network.
Keywords: Cytoplasmic contractions; Fungal growth; Spitzenkörper; Organelle movementRamosa-1Aspergillus nigerNeurospora crassaTrichoderma atroviride
Arthrobacter ardleyensis sp. nov., isolated from Antarctic lake sediment and deep-sea sediment by M. Chen; X. Xiao; P. Wang; X. Zeng; Fengping Wang (pp. 301-305).
Three psychrotrophic Arthrobacter strains, isolated from Antarctic lake sediment (An24, An25T) and deep-sea sediment (ZX6) were studied. Their 16S rRNA gene sequences showed highest similarities (97.0–97.9%) with those of A. nicotianae and A. protophormiae. All three strains underwent rod–coccus morphological change, had high mol% G+C content, were aerobic to slightly anaerobic, and grew between 0°C and 30°C, with optimal growth temperature around 25°C. The cell wall peptidoglycan was A4α variant. DNA–DNA hybridization, physiological and chemotaxonomic studies indicated that these three strains constituted a new homogeneous genomic species within the genus Arthrobacter, for which the name Arthrobacter ardleyensis, with the type strain An25T (CGMCC 1.3685, JCM 12921) was proposed.
Keywords: AntarcticaArthrobacter ardleyensisCell wall; Deep-sea; DNA–DNA hybridization; Mol% G+C content