The Bactericidal Activity of Lectins from Nitrogen-Fixing Bacilli

Lectins I and II isolated from the nitrogen-fixing soil bacterium Paenibacillus polymyxa 1460 were found to be able to suppress the growth of Rhizobium leguminosarum 252 and Bacillus subtilis 36 at nearly all the concentrations tested (from 1 to 10 g/ml). Lectin I was also inhibitory to Azospirillum brasilense 245 and Erwinia carotovora subsp. citrulis 603, while lectin II exerted bactericidal activity against Xanthomonas campestris B-610 and B-611 and A. brasilense 245. The bacillar lectins incubated with Rhizobiumand Azospirillum cells caused leakage of low-molecular-weight substances from the cells, presumably resulting from impairment of the membrane barrier function. We believe that one of the possible mechanisms of the bacterial growth inhibition by lectins is mediated by the lectin-specific receptors occurring on the bacterial membrane, whose interaction with the lectin molecules induces conformational alterations in the membrane and concurrent malfunction of the metabolism of bacterial cells.     

Heteroaromatic monothiocarboxylic acids from Pseudomonas spp

Pyridine derivatives substituted with monothiocarboxylic acid groups are the uniquemetabolites of certain Pseudomonas species. Pyridine-2,6-di-(monothiocarboxylic acid) 1a was found during a screening program for antibiotically active bacterial metabolites due to its ability to complex Fe³⁺. The structure of this complex, itsredox behavior and the biogenesis of the ligand molecule were studied in detail. Thislead to the discovery of a new class of natural products, viz. acylsulfenic acid derivatives. Interest in 1a was revived shortly when complexes with other metals were studied as models for sulfur-containing enzymes. It could also be shown that a quinoline monothiocarboxylic acid derivative acted as an alternative siderophorefor Pseudomonas fluorescens. But a real renaissance was observed only whenthe role of 1a in the degradation of CCl₄ by Pseudomonas stutzeri became evident.     

A specific genetic background is required for acquisition and expression of virulence factors in Escherichia coli

In bacteria, the evolution of pathogenicity seems to be the result of the constant arrival of virulence factors (VFs) into the bacterial genome. However, the integration, retention, and/or expression of these factors may be the result of the interaction between the new arriving genes and the bacterial genomic background. To test this hypothesis, a phylogenetic analysis was done on a collection of 98 Escherichia coli/Shigella strains representing the pathogenic and commensal diversity of the species. The distribution of 17 VFs associated to the different E. coli pathovars was superimposed on the phylogenetic tree. Three major types of VFs can be recognized: (1) VFs that arrive and are expressed in different genetic backgrounds (such as VFs associated with the pathovars of mild chronic diarrhea: enteroaggregative, enteropathogenic, and diffusely-adhering E. coli), (2) VFs that arrive in different genetic backgrounds but are preferentially found, associated with a specific pathology, in only one particular background (such as VFs associated with extraintestinal diseases), and (3) VFs that require a particular genetic background for the arrival and expression of their virulence potential (such as VFs associated with pathovars typical of severe acute diarrhea: enterohemorragic, enterotoxigenic, and enteroinvasive E. coli strains). The possibility of a single arrival of VFs by chance, followed by a vertical transmission, was ruled out by comparing the evolutionary histories of some of these VFs to the strain phylogeny. These evidences suggest that important changes in the genome of E. coli have occurred during the diversification of the species, allowing the virulence factors associated with severe acute diarrhea to arrive in the population. Thus, the E. coli genome seems to be formed by an "ancestral" and a "derived" background, each one responsible for the acquisition and expression of different virulence factors.     

Correlation between bacterial haemoglobin gene (vgb) and aeration: their effect on the growth and alpha-amylase activity in transformed Enterobacter aerogenes

AIMS: To evaluate the effects of bacterial haemoglobin on bacterial growth and alpha-amylase formation under different aeration conditions. METHODS AND RESULTS: Enterobacter aerogenes was transformed with the gene encoding Vitreoscilla (bacterial) haemoglobin, vgb. The growth kinetics and ability to synthesize alpha-amylase enzyme were investigated in this transformed Enterobacter strain as well as in two other Enterobacter control strains that do not harbour the vgb gene. Such comparison was made under variable aeration conditions, using the agitation rate as a measure of aeration. The expression of bacterial haemoglobin-supported cell growth determined as O.D.600 and cell viability in addition to the alpha-amylase production. These positive effects of bacterial haemoglobin were observed under both low and high aerations, but at different extents. CONCLUSIONS: In addition to improving cell growth under low aeration, the bacterial haemoglobin is able to promote bacterial cell tolerance during exposure to high oxygen tension. SIGNIFICANCE AND IMPACT OF THE STUDY: The expression of bacterial haemoglobin is advantageous in reducing the burden of certain toxic conditions such as high oxygen levels. It may have the same impact on some environmental toxic substances. This, haemoglobin biotechnology can be extended to induce enzymes of pollutants degradation or production of some useful industrial substances.     

Listeria monocytogenes mediated CFTR transgene transfer to mammalian cells

Background

Several approaches for gene therapy of cystic fibrosis using viral and non‐viral vectors are currently being undertaken. Nevertheless, the present data suggest that vectors currently being used will either have to be further modified or, alternatively, novel vector systems need to be developed. Recently, bacteria have been proven as suitable vehicles for DNA transfer to a wide variety of eukaryotic cells. In this study, we assessed the ability of the facultative intracellular pathogen Listeria monocytogenes to deliver a cDNA encoding the human cystic fibrosis transmembrane conductance regulator (CFTR) to CHO‐K1 cells, since these cells have been extensively used for heterologous CFTR expression.

Methods

An established in vitro gene transfer system based on antibiotic‐mediated lysis of intracellular L. monocytogenes was exploited to transfer eukaryotic expression plasmids. Transient as well as stable CFTR transgene expression was analyzed by microscopical and biochemical methods; functionality was tested by whole‐cell patch‐clamp recordings.

Results

L. monocytogenes mediated gene transfer to CHO‐K1 cells was facilitated by an improved transfection protocol. In addition, the use of the isogenic mutant L. monocytogenes hlyW491A, engineered to produce a hemolysin variant with low toxigenic activity, greatly enhanced the efficiency of gene transfer. This strain allowed the transfer of functional CFTR to CHO‐K1 cells.

Conclusions

This is the first demonstration of L. monoyctogenes mediated CFTR transgene transfer. The successful in vitro transfer suggests that L. monocytogenes might be a potential vector for cystic fibrosis gene therapy or alternative applications and deserves further investigation in vitro as well as in vivo. Copyright © 2002 John Wiley & Sons, Ltd.

     

Selection of microbial consortia for treating metal-working fluids

The aim of this research was to determine the effectiveness of a strategy for constructing microbial consortia for treating chemically mixed industrial effluent, based on a more thorough understanding of communities within waste metal-working fluids (MWFs). Complementary phenotypic and genotypic methods revealed that the microbial communities in spent MWFs had low diversity and were very similar in species composition in samples originating from different locations and uses. Of 65 bacterial isolates studied, only 9 species were identified using fatty acid methyl ester (FAME) analysis. The results of genotypic analysis by denaturing gradient gel electrophoresis (DGGE) were congruent with observations made using FAME analysis. The metabolic potential of the isolates was assessed in terms of assimilation ability and tolerance of co-contaminants. The three isolates, selected (Clavibacter michiganensis, Methylobacterium mesophilicum, and Rhodococcus erythropolis) to form a consortium, were representative of three of the four most abundant populations and when combined could utilise'or tolerate all of the individual MWF components, including the biocide and the recalcitrant compound benzotriazole. Journal of Industrial Microbiology & Biotechnology (2002) 29, 20–27 doi:10.1038/sj.jim.7000271 Received 19 December 2001/ Accepted in revised form 02 May 2002     

Molecular characterization and estimation of cellulolytic potential of gut bacteria isolated from four white grub species native to Indian Himalayas

An investigation was carried out to isolate, identify and molecularly characterize the cellulose-degrading bacterial isolates from the guts of four white grub species (Anomala bengalensis, Brahmina coriacea, Holotrichia longipennis and Holotrichia setticollis) native to Uttarakhand, Himalayas through 16S rRNA sequencing. A total of 178 bacterial strains were isolated from different gut compartments of selected white grub species, of which 95 bacterial isolates showed cellulose metabolizing activities in the CMC assay. Maximum degraders i.e., 38 were isolated from A. bengalensis, of which 18 were isolated from the fermentation chamber. The value of cellulolytic index ranged between 0.05 and 16 showing a variable cellulolytic activity by degraders. A total of 25 potent strains of cellulose-degrading bacteria recording cellulolytic activity > 1 were isolated and sequenced for 16S rRNA gene. Bacillus stratosphericus strain CBG4MG1 (10.78 ± 4.18), Bacillus cereus strain CBG2FC1 (10.33 ± 3.53), Bacillus sp. strain CBG3MG2 (7.28 ± 0.16) and Paenibacillus ginsengagri strain CBG1FC2 (5.66 ± 2.67) were the most potent cellulose-degrading bacteria isolated from the gut of B. coriacea, H. longipennis, H. setticollis and A. bengalensis, respectively. Thus, the cellulolytic bacteria isolated from the gut of selected white grub species may be good sources for profiling novel isolates for industrial use besides identifying eco-friendly solutions for agro-waste management.     

Increased production of cadaverine and anabasine in hairy root cultures of Nicotiana tabacum expressing a bacterial lysine decarboxylase gene

Several hairy root cultures of Nicotiana tabacum varieties, carrying two direct repeats of a bacterial lysine decarboxylase (ldc) gene controlled by the cauliflower mosaic virus (CaMV) 35S promoter expressed LDC activity up to 1 pkat/mg protein. Such activity was, for example, sufficient to increase cadaverine levels of the best line SR3/1-K1,2 from ca. 50 g (control cultures) to about 700 g/g dry mass. Some of the overproduced cadaverine of this line was used for the formation of anabasine, as shown by a 3-fold increase of this alkaloid. In transgenic lines with lower LDC activity the changes of cadaverine and anabasine levels were correspondingly lower and sometimes hardly distinguishable from controls. Feeding of lysine to root cultures, even to those with low LDC activity, greatly enhanced cadaverine and anabasine livels, while the amino acid had no or very little effect on controls and LDC-negative lines.     

驯化噬菌体提高噬菌体对碳青霉烯类耐药肺炎克雷伯菌的杀菌能力

【目的】本研究旨在通过驯化提高噬菌体的裂解能力并降低其宿主菌耐受性产生的速度,从而提高对重要病原菌-碳青霉烯类耐药肺炎克雷伯菌(carbapenem-resistant Klebsiella pneumoniae, CRKp)的杀菌效果。【方法】以临床CRKp菌株Kp2092为宿主菌,利用双层琼脂平板法从污水中分离噬菌体并分析其裂解谱;对其中的广谱强裂解性噬菌体通过透射电镜观察其形态特征并进行全基因组测序;通过噬菌体-宿主连续培养进行噬菌体驯化,并比较驯化前后噬菌体生物学特性的差异。【结果】分离得到的9株肺炎克雷伯菌噬菌体中,噬菌体P55anc裂解能力强且裂解谱广,透射电镜观察发现其为短尾噬菌体。P55anc基因组全长40 301 bp,包含51个编码序列,其中27个具有已知功能,主要涉及核酸代谢、噬菌体结构蛋白、DNA包装和细胞裂解等。噬菌体P55anc经9 d的驯化后,得到3株驯化噬菌体。驯化后噬菌体杀菌能力增强,主要表现为细菌生长曲线显著下降、噬菌体暴发量增多、裂解谱扩大,且宿主菌对其产生抗性的概率显著降低。与此同时,驯化后的噬菌体在热处理、紫外暴露以及血清等环境下保持较好的稳定性。【结论】利用噬菌体-宿主连续培养的方法可对噬菌体进行驯化和筛选,驯化后的噬菌体杀菌效果更强,且在不同压力处理下的稳定性良好,而细菌产生噬菌体抗性的概率也降低。