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Archives of Microbiology (v.194, #12)

Cell wall active antibiotics reduce chromosomal DNA fragmentation by peptidoglycan hydrolysis in Staphylococcus aureus by María Tamayo; Rebeca Santiso; Jaime Gosálvez; Germán Bou; María del Cármen Fernández; José Luis Fernández (pp. 967-975).

Lysostaphin digestion of peptidoglycan (PG) from Staphylococcus aureus resulted in chromosomal DNA fragmentation by released DNase, as directly visualized in situ on isolated nucleoids. Nevertheless, DNA digestion was partially prevented by previous incubation with antibiotics that inhibit PG synthesis. This inhibitory effect was much more remarkable with glycopeptides vancomycin and mainly teicoplanin than with beta-lactams cloxacillin and ceftazidime. Therefore, inhibition of PG chain elongation has a more significant inhibition of DNA degradation than inhibition of PG cross-linking, possibly due to a reduction in DNase storage at the cell wall.

Keywords: DNA fragmentation; DNase; Staphylococcus aureus ; Glycopeptides

FliA expression analysis and influence of the regulatory proteins RpoN, FleQ and FliA on virulence and in vivo fitness in Legionella pneumophila by Tino Schulz; Kerstin Rydzewski; Eva Schunder; Gudrun Holland; Norbert Bannert; Klaus Heuner (pp. 977-989).

In Legionella pneumophila, the regulation of the flagellum and the expression of virulence traits are linked. FleQ, RpoN and FliA are the major regulators of the flagellar regulon. We demonstrated here that all three regulatory proteins mentioned (FleQ, RpoN and FliA) are necessary for full in vivo fitness of L. pneumophila strains Corby and Paris. In this study, we clarified the role of FleQ for fliA expression from the level of mRNA toward protein translation. FleQ enhanced fliA expression, but FleQ and RpoN were not necessary for basal expression. In addition, we identified the initiation site of fliA in L. pneumophila and found a putative σ⁷⁰ promoter element localized upstream. The initiation site was not influenced in the ΔfleQ or ΔrpoN mutant strain. We demonstrated that there is no significant difference in the regulation of fliA between strains Corby and Paris, but the FleQ-dependent induction of fliA transcription in the exponential phase is stronger in strain Paris than in strain Corby. In addition, we showed for the first time the presence of a straight hook at the pole of the non-flagellated ΔfliA and ΔfliD mutant strains by electron microscopy, indicating the presence of an intact basal body in these strains.

Keywords: Legionella pneumophila ; Paris; Corby; Flagellin regulation; FleQ; FliA; RpoN; Virulence; In vivo fitness; Straight hook

Study of the influence of sporulation conditions on heat resistance of Geobacillus stearothermophilus used in the development of biological indicators for steam sterilization by Belquis P. Guizelini; Luciana P. S. Vandenberghe; Sandra Regina B. R. Sella; Carlos Ricardo Soccol (pp. 991-999).

Biological indicators are important tools in infection control via sterilization process monitoring. The use of a standardized spore crop with a well-defined heat resistance will guarantee the quality of a biological indicator. Ambient factors during sporulation can affect spore characteristics and properties, including heat resistance. The aim of this study is to evaluate the main sporulation factors responsible for heat resistance in Geobacillus stearothermophilus, a useful biological indicator for steam sterilization. A sequence of a three-step optimization of variables (initial pH, nutrient concentration, tryptone, peptone, beef extract, yeast extract, manganese sulfate, magnesium sulfate, calcium chloride and potassium phosphate) was carried out to screen those that have a significant influence on heat resistance of produced spores. The variable exerting greatest influence on G. stearothermophilus heat resistance during sporulation was found to be the initial pH. Lower nutrient concentration and alkaline pH around 8.5 tended to enhance decimal reduction time at 121 °C (D_121°C). A central composite design enabled a fourfold enhancement in heat resistance, and the model obtained accurately describes positive pH and negative manganese sulfate concentration influence on spore heat resistance.

Keywords: Biological indicator; Geobacillus stearothermophilus ; Response surface methodology; Steam sterilization

Study on diversity of endophytic bacterial communities in seeds of hybrid maize and their parental lines by Yang Liu; Shan Zuo; Liwen Xu; Yuanyuan Zou; Wei Song (pp. 1001-1012).

The seeds of plants are carriers of a variety of beneficial bacteria and pathogens. Using the non-culture methods of building 16S rDNA libraries, we investigated the endophytic bacterial communities of seeds of four hybrid maize offspring and their respective parents. The results of this study show that the hybrid offspring Yuyu 23, Zhengdan958, Jingdan 28 and Jingyu 11 had 3, 33, 38 and 2 OTUs of bacteria, respectively. The parents Ye 478, Chang 7-2, Zheng 58, Jing 24 and Jing 89 had 12, 36, 6, 12 and 2 OTUs, respectively. In the hybrid Yuyu 23, the dominant bacterium Pantoea (73.38 %) was detected in its female parent Ye 478, and the second dominant bacterium of Sphingomonas (26.62 %) was detected in both its female (Ye 478) and male (Chang 7-2) parent. In the hybrid Zhengdan 958, the first dominant bacterium Stenotrophomonas (41.67 %) was detected in both the female (Zheng 58) and male (Chang 7-2) parent. The second dominant bacterium Acinetobacter (9.26 %) was also the second dominant bacterium of its male parent. In the hybrid Jingdan 28, the second dominant bacterium Pseudomonas (12.78 %) was also the second dominant bacterium of its female parent, and its third dominant bacterium Sphingomonas (9.90 %) was the second dominant bacterium of its male parent and detected in its female parent. In the hybrid Jingyu 11, the first dominant bacterium Leclercia (73.85 %) was the third dominant bacterium of its male parent, and the second dominant bacterium Enterobacter (26.15 %) was detected in its male parent. As far as we know, this was the first research reported in China on the diversity of the endophytic bacterial communities of the seeds of various maize hybrids with different genotypes.

Keywords: Hybrid maize; Seed endophytic bacteria; Bacterial diversity; Culture-independent method

The antibiosis of nodule-endophytic agrobacteria and its potential effect on nodule functioning of Phaseolus vulgaris by Seif-Allah Chihaoui; Haythem Mhadhbi; Ridha Mhamdi (pp. 1013-1021).

The effect of the nodule-endophytic Agrobacterium strain 10C2 on nodulation, plant growth and nodule functioning of Phaseolus vulgaris was investigated using two rhizobial strains differing in their sensitivity to the in vitro antibiosis of the Agrobacterium strain. In the case of the sensitive strain, Agrobacterium sp. 10C2 induced a significant decrease in the proportion of pink nodules, probably by an antibiosis effect leading to the reduction in the number of bacteroids and thereby a decrease in total soluble proteins, leghaemoglobin content, photosynthesis and nitrogen fixation. In this case, the Agrobacterium strain behaved like a plant pathogen and the nodule reacted by increasing guaiacol peroxidase (POX) activity, which assures some physiological processes linked to pathogen control. By contrast, in the case of the resistant strain, the proportion of pink nodules increased, and thereby total soluble proteins, leghaemoglobin content, biomass production and nitrogen fixation were enhanced. The Agrobacterium strain is regarded in this case as a plant growth–promoting rhizobacterium and the POX-pathogen reaction was not observed. There was even a decrease in superoxide dismutase activity. The results suggested also that the Agrobacterium strain may be also involved in retarding nodule senescence in the case of the resistant strain.

Keywords: Agrobacterium ; Antioxidant enzymes; Endophyte; Nitrogen fixation; Nodule senescence; Rhizobium

Knockout of fatty acid desaturase genes in Pichia pastoris GS115 and its effect on the fatty acid biosynthesis and physiological consequences by Ai-Qun Yu; Jian-Chun Zhu; Biao Zhang; Lai-Jun Xing; Ming-Chun Li (pp. 1023-1032).

Unsaturated fatty acids (UFAs), including oleic acid (OA, C18:1n-9), linoleic acid (LA, C18:2n-6) and α-linolenic acid (ALA, C18:3n-3), are major components of membrane lipids in Pichia pastoris GS115. In order to clarify the biosynthesis pathway of UFAs on the molecular level and investigate their possible roles in growth and development of this strain, we here report modified strains with disrupted desaturase gene by homologous recombination. Gas chromatography analysis of fatty acid composition in the corresponding mutants confirmed that ∆¹²-desaturase encoded by Fad12 was responsible for the formation of LA, and ALA was synthesized by ∆¹⁵-desaturase encoded by Fad15. Simultaneous deletion of Fad9A and Fad9B was lethal and supplementation of OA could restore growth, indicating that possibly both Fad9A and Fad9B encoded ∆⁹-desaturase that converted SA into OA. Phenotypic analysis demonstrated that wild type and Fad15 mutant grew at almost the same rate, Fad12 mutant grew much slower than these two strains. Moreover, OA was positively correlated to cold tolerance and ethanol tolerance of GS115, whereas LA and ALA did not affect cold tolerance and ethanol tolerance of it. In addition, we showed that tolerance of GS115 to high concentration of methanol was independent of these three UFAs.

Keywords: P. pastoris ; Fad9A ; Fad9B ; Fad12 ; Fad15 ; Gene disruption

Impairment of ribosomal subunit synthesis in aminoglycoside-treated ribonuclease mutants of Escherichia coli by Ashley D. Frazier; W. S. Champney (pp. 1033-1041).

The bacterial ribosome is an important target for many antimicrobial agents. Aminoglycoside antibiotics bind to both 30S and 50S ribosomal subunits, inhibiting translation and subunit formation. During ribosomal subunit biogenesis, ribonucleases (RNases) play an important role in rRNA processing. E. coli cells deficient for specific processing RNases are predicted to have an increased sensitivity to neomycin and paromomycin. Four RNase mutant strains showed an increased growth sensitivity to both aminoglycoside antibiotics. E. coli strains deficient for the rRNA processing enzymes RNase III, RNase E, RNase G or RNase PH showed significantly reduced subunit amounts after antibiotic treatment. A substantial increase in a 16S RNA precursor molecule was observed as well. Ribosomal RNA turnover was stimulated, and an enhancement of 16S and 23S rRNA fragmentation was detected in E. coli cells deficient for these enzymes. This work indicates that bacterial RNases may be novel antimicrobial targets.

Keywords: Escherichia coli ; Ribonuclease mutants; Ribosome assembly; Neomycin; Paromomycin

Isolation and characterization of acyl homoserine lactone–producing bacteria during an urban river biofilm formation by Yili Huang; Jing Zhang; Zhiliang Yu; Yanhua Zeng; Yuqi Chen (pp. 1043-1048).

The presence and diversity of acyl homoserine lactone (AHL)-producers in an urban river biofilm were investigated during 60-day biofilm formation. AHL biosensors detected the presence of AHL-producers in 1–60-day river biofilms. Screening for AHL-producers resulted in 17 Aeromonas spp., 3 Pseudomonas spp., 3 Ensifer spp., and 1 Acinetobacter sp. Among these isolates, six of them were closely related to Acinetobacter tjernbergiae, Aeromonas allosaccharophila, Aeromonas aquariorum, Aeromonas jandaei, Pseudomonas panipatensis, and Ensifer adhaerens and represented novel AHL-producing species. Thin layer chromatography revealed that C4-homoserine lactone was prevailing in Aeromonas spp., whereas C6- and C8-homoserine lactones and their derivatives were prevailing in other strains. Using degenerate primers, novel AHL synthetase genes from the three Ensifer spp. were successfully amplified. This study reports for the first time the diversity of AHL-producers from a river biofilm and the variety of novel AHL synthetase genes in Ensifer group.

Keywords: River biofilm; Acyl homoserine lactone; Aeromonas ; Ensifer

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Archives of Microbiology (v.194, #12)