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Archives of Microbiology (v.194, #8)

Uridylylation of Herbaspirillum seropedicae GlnB and GlnK proteins is differentially affected by ATP, ADP and 2-oxoglutarate in vitro by Ana C. Bonatto; Emanuel M. Souza; Marco A. S. Oliveira; Rose A. Monteiro; Leda S. Chubatsu; Luciano F. Huergo; Fábio O. Pedrosa (pp. 643-652).

PII are signal-transducing proteins that integrate metabolic signals and transmit this information to a large number of proteins. In proteobacteria, PII are modified by GlnD (uridylyltransferase/uridylyl-removing enzyme) in response to the nitrogen status. The uridylylation/deuridylylation cycle of PII is also regulated by carbon and energy signals such as ATP, ADP and 2-oxoglutarate (2-OG). These molecules bind to PII proteins and alter their tridimensional structure/conformation and activity. In this work, we determined the effects of ATP, ADP and 2-OG levels on the in vitro uridylylation of Herbaspirillum seropedicae PII proteins, GlnB and GlnK. Both proteins were uridylylated by GlnD in the presence of ATP or ADP, although the uridylylation levels were higher in the presence of ATP and under high 2-OG levels. Under excess of 2-OG, the GlnB uridylylation level was higher in the presence of ATP than with ADP, while GlnK uridylylation was similar with ATP or ADP. Moreover, in the presence of ADP/ATP molar ratios varying from 10/1 to 1/10, GlnB uridylylation level decreased as ADP concentration increased, whereas GlnK uridylylation remained constant. The results suggest that uridylylation of both GlnB and GlnK responds to 2-OG levels, but only GlnB responds effectively to variation on ADP/ATP ratio.

Keywords: PII proteins; GlnB; GlnK; Uridylylation; Herbaspirillum seropedicae

Hydrolysis of fungal and plant cell walls by enzymatic complexes from cultures of Fusarium isolates with different aggressiveness to rye (Secale cereale) by Jolanta Jaroszuk-Ściseł; Ewa Kurek (pp. 653-665).

The efficiency of hydrolysis of fungal (Fusarium spp.) cell wall and rye root cell wall by crude enzymatic complexes from (42-day-old) cultures of three F. culmorum isolates, a plant growth–promoting rhizosphere isolate (PGPF) DEMFc2, a deleterious rhizosphere isolate (DRMO) DEMFc5, and a pathogenic isolate DEMFc37, as well as two other, pathogenic isolates belonging to F. oxysporum and F. graminearum species was studied. In the enzymatic complexes originating from the Fusarium spp. cultures, the activities of the following cell wall–degrading enzymes were identified: glucanases, chitinases, xylanases, endocellulases, exocellulases, pectinases, and polygalacturonases. The preparation originating from a culture of the PGPF isolate was the least efficient in plant cell wall (PCW) hydrolysis. There were no significant differences in the efficiency of PCW hydrolysis between preparations from cultures of the DRMO and the pathogenic isolates. PGPF was the most efficient in liberating reducing sugars and N-acetylglucosamine (GlcNAc) from fungal cell walls (FCW). Xylanase activities of the enzymatic complexes were strongly positively (R > +0.9) correlated with their efficiency in hydrolyzing PCW, whereas chitinase activities were correlated with the efficiency in FCW hydrolysis.

Keywords: Efficiency of cell wall hydrolysis; PGPF; DRMO; Pathogenic fungi; Xylanase activity; Chitinase activity

Patterns of sheath elongation, cell proliferation, and manganese(II) oxidation in Leptothrix cholodnii by Minoru Takeda; Yuta Kawasaki; Takuto Umezu; Shoichi Shimura; Makoto Hasegawa; Jun-ichi Koizumi (pp. 667-673).

Leptothrix cholodnii is a Mn(II)-oxidizing and sheath-forming member of the class β-Proteobacteria. Its sheath is a microtube-like filament that contains a chain of cells. From a chemical perspective, the sheath can be described as a supermolecule composed of a cysteine-rich polymeric glycoconjugate, called thiopeptidoglycan. However, the mechanism that controls the increase in sheath length is unknown. In this study, we attempted to detect sheath elongation through microscopic examination by using conventional reagents. Selective fluorescent labeling of preexisting or newly formed regions of the sheath was accomplished using combinations of biotin-conjugated maleimide, propionate-conjugated maleimide, and a fluorescent antibiotin antibody. Epifluorescence microscopy indicated that the sheath elongates at the terminal regions. On the bases of this observation, we assumed that the newly secreted thiopeptidoglycan molecules are integrated into the preexisting sheath at its terminal ends. Successive phase-contrast microscopy revealed that all cells proliferate at nearly the same rate regardless of their positions within the sheath. Mn(II) oxidation in microcultures was also examined with respect to cultivation time. Results suggested that the deposition of Mn oxides is notable in the aged regions. The combined data reveal the spatiotemporal relationships among sheath elongation, cell proliferation, and Mn oxide deposition in L. cholodnii.

Keywords: Leptothrix cholodnii ; Sheath; Elongation; Cell proliferation; Mn(II) oxidation

Secretome analysis of Clostridium difficile strains by Alexander Boetzkes; Katharina Wiebke Felkel; Johannes Zeiser; Nelli Jochim; Ingo Just; Andreas Pich (pp. 675-687).

Clostridium difficile causes infections ranging from mild C. difficile-associated diarrhea to severe pseudomembranous colitis. Since 2003 new hypervirulent C. difficile strains (PCR ribotype 027) emerged characterized by a dramatically increased mortality. The secretomes of the three C. difficile strains CDR20291, CD196, and CD630 were analyzed and compared. Proteins were separated and analyzed by means of SDS--PAGE and LC–MS. MS data were analyzed using Mascot and proteins were checked for export signals with SecretomeP and SignalP. LC–MS analysis revealed 158 different proteins in the supernatant of C. difficile. Most of the identified proteins originate from the cytoplasm. Thirty-two proteins in CDR20291, 36 in CD196 and 26 in CD630 were identified to be secreted by C. difficile strains. Those were mainly S-layer proteins, substrate-binding proteins of ABC-transporters, cell wall hydrolases, pilin and unknown hypothetical proteins. Toxin A and toxin B were identified after growth in brain heart infusion medium using immunological techniques. The ADP-ribosyltransferase-binding component protein, which is a part of the binary toxin CDT, was only identified in the hypervirulent ribotype 027 strains. Further proteins that are secreted specifically by hypervirulent strains were identified.

Keywords: Clostridium difficile ; Hypervirulent strains; Secretome; MS analysis

Indigenous hydrocarbon-utilizing bacterioflora in oil-polluted habitats in Kuwait, two decades after the greatest man-made oil spill by H. Al-Awadhi; D. Al-Mailem; N. Dashti; M. Khanafer; S. Radwan (pp. 689-705).

Kuwaiti habitats with two-decade history of oil pollution were surveyed for their inhabitant oil-utilizing bacterioflora. Seawater samples from six sites along the Kuwaiti coasts of the Arabian Gulf and desert soil samples collected from seven sites all over the country harbored oil-utilizing bacteria whose numbers made up 0.0001–0.01% of the total, direct, microscopic counts. The indigenous bacterioflora in various sites were affiliated to many species. This was true when counting was made on nitrogen-containing and nitrogen-free media. Seawater samples harbored species belonging predominantly to the Gammaproteobacteria and desert soil samples contained predominantly Actinobacteria. Bacterial species that grew on the nitrogen-free medium and that represented a considerable proportion of the total in all individual bacterial consortia were diazotrophic. They gave positive acetylene-reduction test and possessed the nifH genes in their genomes. Individual representative species could utilize a wide range of aliphatic and aromatic hydrocarbons, as sole sources of carbon and energy. Quantitative determination showed that the individual species consumed crude oil, n-octadecane and phenanthrene, in batch cultures. It was concluded that the indigenous microflora could be involved in bioremediation programs without bioaugmentation or nitrogen fertilization. Irrigation would be the most important practice in bioremediation of the polluted soil desert areas.

Keywords: Bioremediation; Desert soil; Diazotrophs; Hydrocarbons; Oil pollution

qPCR quantification of Sphaerodes mycoparasitica biotrophic mycoparasite interaction with Fusarium graminearum: in vitro and in planta assays by Vladimir Vujanovic; Yit Kheng Goh (pp. 707-717).

Sphaerodes mycoparasitica, a biotrophic mycoparasite of Fusarium species, improved wheat seed germination and seedling growth in vitro compared to Trichoderma harzianum, a necrotrophic mycoparasite. However, under phytotron conditions, both S. mycoparasitica and T. harzianum had positive impact on wheat seedlings growth in the presence of F. graminearum. Once exposed to the mycoparasites, the DNA quantity of F. graminearum in wheat root decreased. Observed shifts in DNA quantity using qPCR, a set of newly designed Sphaerodes-specific SmyITS primers, as well as Trichoderma-TGP4 and Fusarium-Fg16 N primers, demonstrated the mycoparasite’s biocontrol effectiveness in planta. In the presence of F. graminearum, the concentration of S. mycoparasitica DNA remained stable in the root, whereas the amount of T. harzianum DNA decreased. The toxicity assays indicated that S. mycoparasitica’s mycelia withstand higher concentrations of deoxynivalenol, 3-acetyldeoxynivalenol, and zearalenone mycotoxins than T. harzianum mycelia. This study compares the ability of two fungi to improve the wheat growth, decrease the root colonization of Fusarium, and withstand mycotoxins.

Keywords: Fusarium graminearum ; Sphaerodes ; Trichoderma ; Mycoparasites; qPCR; ITS rDNA-specific primers; Mycotoxins

Gallium(III), cobalt(III) and copper(II) protoporphyrin IX exhibit antimicrobial activity against Porphyromonas gingivalis by reducing planktonic and biofilm growth and invasion of host epithelial cells by Teresa Olczak; Dorota Maszczak-Seneczko; John W. Smalley; Mariusz Olczak (pp. 719-724).

Porphyromonas gingivalis acquires heme for growth, and initiation and progression of periodontal diseases. One of its heme acquisition systems consists of the HmuR and HmuY proteins. This study analyzed the antimicrobial activity of non-iron metalloporphyrins against P. gingivalis during planktonic growth, biofilm formation, epithelial cell adhesion and invasion, and employed hmuY, hmuR and hmuY-hmuR mutants to assess the involvement of HmuY and HmuR proteins in the acquisition of metalloporphyrins. Iron(III) mesoporphyrin IX (mesoheme) and iron(III) deuteroporphyrin IX (deuteroheme) supported planktonic growth of P. gingivalis cells, biofilm accumulation, as well as survival, adhesion and invasion of HeLa cells in a way analogous to protoheme. In contrast, cobalt(III), gallium(III) and copper(II) protoporphyrin IX exhibited antimicrobial activity against P. gingivalis, and thus represent potentially useful antibacterial compounds with which to target P. gingivalis. P. gingivalis hmuY, hmuR and hmuY-hmuR mutants showed decreased growth and infection of epithelial cells in the presence of all metalloporphyrins examined. In conclusion, the HmuY protein may not be directly involved in transport of free metalloporphyrins into the bacterial cell, but it may also play a protective role against metalloporphyrin toxicity by binding an excess of these compounds.

Keywords: Porphyromonas gingivalis ; HmuY; Non-iron metalloporphyrins; Heme; Biofilm; Epithelial cell invasion

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Archives of Microbiology (v.194, #8)