Molecular analysis of a consortium of ruminal microbes that detoxify pyrrolizidine alkaloids

Members of a consortium of bacteria, isolated from the rumen of sheep, that degrades pyrrolizidine alkaloids (PAs) found in tansy ragwort (Senecio jacobaea) were characterized. An enrichment of ruminal bacteria was isolated from a sample of ruminal fluid using standard anaerobic techniques. The PA degradative capacity of the enrichment was tested by spiking purified PA extract from tansy ragwort. Length heterogeneity analysis by PCR (LH-PCR) and restriction fragment length polymorphism (RFLP) analysis was used to identify members of the consortium. Phylogenetic analysis of the 16S rDNA gene revealed differing results based on the molecular method used. LH-PCR identified 7 different organisms in 3 groups while RFLP identified 6 organisms with differing banding patterns in 5 groups. After the phylogenetic analyses of both methods were combined, the combined isolates represented 6 groups. The majority of the members of this consortium are <97.0% homologous with known bacteria, indicating this consortium may contain novel organisms able to detoxify PAs found in tansy ragwort. Further understanding of the metabolic pathways used by this consortium to degrade PAs could lead to the use of the consortium as a probiotic therapy for livestock and horses afflicted with tansy ragwort toxicosis.     

Molecular cross-talk between sponge host and associated microbes

Marine organisms especially those that live sessile, as sponges, are well known to have specific relationships with a great variety of microorganisms including bacteria and fungi. As most simple metazoan phylum, the Porifera, which emerged first during the transition from the non-Metazoa to the Metazoa from the common ancestor, comprise wide arrays of recognition molecules, both for Gram-negative bacteria and for Gram-positive bacteria as well as for fungi. They react specifically with effector molecules to inhibit or kill the invading microorganisms. The elicitation and the subsequent effector reactions of the sponges towards these microbes are outlined. However, besides of the elimination of bacteria and fungi, some of those taxa are kept as symbionts of the sponges, allowing them, for example, to accumulate the essential element manganese or to synthesize carotinoids. The sponges produce low-molecular-weight bioactive compounds, secondary metabolites, to eliminate the microorganisms. In addition, they are armed with cationic antimicrobial peptides allowing them to defend against invasive microorganisms and, in parallel, to kill or repel also metazoan invaders. The broad range of chemically and functionally different compounds qualifies the Porifera as the most important animal phylum to be exploited as a source for the isolation of new potential drugs. First molecular biological strategies have been outlined to obtain those compounds in a sustainable way, by producing them recombinantly.     

Dielectrophoretic analysis of microbes in water

Traditional microbiological methods are still used extensively for analysis of micro-organisms in water. However, they are inefficient due to a high labour input requirement, a low sample capacity, and often a long time lag before results are available. Analytical stages involving incubation and growth (enrichments and colony isolation) contribute the greatest delay in reporting, although subsequent identification can also be protracted. The use of electrometric growth analysers (measuring impedance, conductance or capacitance changes) is now more common in water microbiology. Although these instruments can provide more rapid results and provide increased handling capacity, the bacterial generation times required to provide detectable changes cause delays and suitable selective media are not fully developed for all microbes of interest. Most other recent methods have equally disappointing drawbacks and thus extensive research continues in order to realise the ambition of 'real-time' analytical microbiology. Several research groups have demonstrated the potential of dielectrophoresis in providing microbial concentration, separation and identification systems which are not limited by bacterial growth and are therefore extremely rapid. Dielectrophoresis occurs when cells are placed in non-uniform electric fields. The cells move towards the electrodes (regardless of the direction of the applied field) as determined by their dielectric properties (conductivity and permittivity) rather than by their charge as occurs in electrophoresis. Also, the polarisability of the cells, and therefore the polarity and magnitude of the dielectrophoretic force, varies as a function of the electric field frequency. Because the dielectric properties of a particular cell type have characteristic frequency-dependent components, if cell collection at electrodes is observed across a frequency range, the collection spectrum produced is distinctive for the cell type under investigation. This can be exploited for analytical and separation applications in microbiology. This paper will describe rapid analytical techniques based on electrokinetic phenomena under research and development at York. These include dielectrophoretic enrichment, concentration and characterisation systems for the analysis of water bacteria and protozoa.     

Molecular survey of concrete sewer biofilm microbial communities

The microbial composition of concrete biofilms within wastewater collection systems was studied using molecular assays. SSU rDNA clone libraries were generated from 16 concrete surfaces of manholes, a combined sewer overflow, and sections of a corroded sewer pipe. Of the 2457 sequences analyzed, α-, β-, γ-, and δ-Proteobacteria represented 15%, 22%, 11%, and 4% of the clones, respectively. β-Proteobacteria (47%) sequences were more abundant in the pipe crown than any of the other concrete surfaces. While 178 to 493 Operational Taxonomic Units (OTUs) were associated with the different concrete samples, only four sequences were shared among the different clone libraries. Bacteria implicated in concrete corrosion were found in the clone libraries while archaea, fungi, and several bacterial groups were also detected using group-specific assays. The results showed that concrete sewer biofilms are more diverse than previously reported. A more comprehensive molecular database will be needed to better study the dynamics of concrete biofilms.     

Molecular survey of concrete sewer biofilm microbial communities

The microbial composition of concrete biofilms within wastewater collection systems was studied using molecular assays. SSU rDNA clone libraries were generated from 16 concrete surfaces of manholes, a combined sewer overflow, and sections of a corroded sewer pipe. Of the 2457 sequences analyzed, α-, β-, γ-, and δ-Proteobacteria represented 15%, 22%, 11%, and 4% of the clones, respectively. β-Proteobacteria (47%) sequences were more abundant in the pipe crown than any of the other concrete surfaces. While 178 to 493 Operational Taxonomic Units (OTUs) were associated with the different concrete samples, only four sequences were shared among the different clone libraries. Bacteria implicated in concrete corrosion were found in the clone libraries while archaea, fungi, and several bacterial groups were also detected using group-specific assays. The results showed that concrete sewer biofilms are more diverse than previously reported. A more comprehensive molecular database will be needed to better study the dynamics of concrete biofilms.     

Molecular mechanisms involved in Bacillus subtilis biofilm formation

Biofilms are the predominant lifestyle of bacteria in natural environments, and they severely impact our societies in many different fashions. Therefore, biofilm formation is a topic of growing interest in microbiology, and different bacterial models are currently studied to better understand the molecular strategies that bacteria undergo to build biofilms. Among those, biofilms of the soil‐dwelling bacterium Bacillus subtilis are commonly used for this purpose. Bacillus subtilis biofilms show remarkable architectural features that are a consequence of sophisticated programmes of cellular specialization and cell–cell communication within the community. Many laboratories are trying to unravel the biological role of the morphological features of biofilms, as well as exploring the molecular basis underlying cellular differentiation. In this review, we present a general perspective of the current state of knowledge of biofilm formation in B. subtilis and thereby placing a special emphasis on summarizing the most recent discoveries in the field.     

Molecular analysis and development of 16S rRNA oligonucleotide probes to characterize a diclofop-methyl-degrading biofilm consortium

Genomic DNA from nine individual bacteria, isolated from a diclofop-methyl-degrading biofilm consortium, was extracted for genetic characterization. The degradation of diclofop-methyl produces metabolites that are known intermediates or substrates for bacteria that degrade a variety of chlorinated aromatic compounds. Accordingly, oligonucleotide primers were designed from specific catabolic genes for chlorinated organic degradation pathways, and tested by PCR to determine if these genes are involved in diclofop-methyl degradation. DNA homology between the PCR products and the known catabolic genes investigated by Southern hybridization analysis and by sequencing, suggested that novel catabolic genes are functioning in the isolates. Specific fluorescent oligonucleotides were designed for two of the isolates, following 16S rDNA sequencing and identification of each of the isolates. These probes were successfully used for fluorescent in situ hybridization (FISH) studies of the two isolates in the biofilm consortium.     

Molecular signals in the interactions between plants and microbes.

The field of plant-microbe interactions has witnessed several recent breakthroughs, such as the molecular details of vir gene induction, identification of Nod factors, and the cloning and characterization of avr genes. Other breakthroughs, such as the cloning and characterization of R genes, appear imminent. Parallels to mammalian systems are emerging in the world of plant-microbe interactions, for example, ion channels formed by Rhizobium proteins, similarities of hrp genes to pathogenicity genes of mammalian pathogens, and plant signal transduction via calcium and protein phosphorylation. We remain, however, largely ignorant of many facets of signaling in plant-microbe interactions. We know little about how microbial signals are perceived by plants or how subsequent signal transduction occurs within plant cells and are probably unaware of many of the microbe-generated signals to which plants respond or of plant-generated signals to which bacteria and fungi respond. Contributions from those working on the genetics, molecular biology, and physiology of bacteria, fungi, and plants will be required to address these questions. The many nonpathogenic plant-microbe interactions in addition to the Rhizobium-plant interaction remain relatively unexplored. Genetic and molecular approaches are being initiated to investigate the signaling that is likely to underlie interactions such as those between mycorrhizal fungi and plant roots and between epiphytic bacteria and plant leaf surfaces. The importance of these interactions to plant growth and development makes it likely that they will figure more prominently at future symposia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative analysis of biofilm thickness variability

The thickness variability of biofilms of Pseudomonas aeruginosa, Klebsiella pneumoniae, and the binary population combination of these two species was quantified. The experimental method involved cryoembedding biofilms with a commercial tissue embedding agent, sectioning, and applying image analysis to construct thickness profiles along linear transects (up to 1 cm in length) across the substratum. Biofilms embedded and sectioned by this method were locally as thin as a single cell attached to the surface (<5 mum) and as thick as 1000 mum. Week-old biofilms of three different species compositions displayed distinct structural features as indicated by their mean thicknesses and by a roughness coefficient. Monopopulation biofilms of P. aeruginosa (29 mum mean thickness) or K. pneumoniae (100 mum mean thickness) were thinner than the binary population biofilm (400 mum mean thickness). A roughness coefficient developed in this investigation corroborated the qualitative visual characterization of P. aeruginosa biofilms as relatively uniformly thick (mean roughness coefficient 0.15), K. pneumoniae biofilms as patchy (mean roughness coefficient 1.14), and the binary population biofilm as intermediate (mean roughness coefficient 0.26). Whereas P. aeruginosa and binary population biofilms covered the substratum completely, significant areas of essentially bare substratum were apparent in K. pneumoniae biofilms. The patchiness of K. pneumoniae biofilms may be due to the fact that this organism is nonmotile. A spatial correlation analysis of the thickness data indicated that thickness measurements were still correlated even when separated by distances that exceeded the mean biofilm thickness. Cell aggregates, some of them hundreds of microns in size, were observed in the effluent of K. pneumoniae and binary population biofilm reactors. Measurements of thickness variability and other observations reported in this article provide a quantitative basis for analysis of microscale structural heterogeneity of biofilms. (c) 1995 John Wiley & Sons, Inc.     

Discovery of commonly existing anode biofilm microbes in two different wastewater treatment MFCs using FLX Titanium pyrosequencing

In microbial fuel cells (MFC), wastewater is used as a fuel while organic and nutrient pollution in the wastewater are being treated. In the present study, commonly existing microbial populations in MFC anode biofilms were identified using high throughput FLX Titanium pyrosequencing to provide much more extensive information of anode microbial communities than previously possible. Using 454 FLX Titanium pyrosequencing, 31,901 sequence reads with an average length of 430 bp were obtained from 16S rRNA gene amplicons from different MFC anodes with different substrate exposure and respiration conditions, and microbial community structure and population identification were then analyzed using high-throughput bioinformatics methods. Although community profiles from the four samples were significantly different, hierarchical clustering analysis revealed several bacterial populations that commonly exist in the anode biofilm samples. These bacteria were phylogenetically distributed in Firmicutes and the alpha-, beta-, gamma-, and delta-subclasses of Proteobacteria. In addition, most of these populations were found to be novel anode bacteria and exhibited oligotrophic or substrate-concentration-insensitive growth. These findings suggest that commonly existing anode bacteria may play a key role in the stable operations of MFCs, combined with wastewater treatment plants, under fluctuating substrate and respiration conditions.     

Molecular mechanisms of compounds affecting bacterial biofilm formation and dispersal

Bacteria can switch between planktonic forms (single cells) and biofilms, i.e., bacterial communities growing on solid surfaces and embedded in a matrix of extracellular polymeric substance. Biofilm formation by pathogenic bacteria often results in lower susceptibility to antibiotic treatments and in the development of chronic infections; thus, biofilm formation can be considered an important virulence factor. In recent years, much attention has been directed towards understanding the biology of biofilms and towards searching for inhibitors of biofilm development and of biofilm-related cellular processes. In this report, we review selected examples of target-based screening for anti-biofilm agents: We focus on inhibitors of quorum sensing, possibly the most characterized target for molecules with anti-biofilm activity, and on compounds interfering with the metabolism of the signal molecule cyclic di-GMP metabolism and on inhibitors of DNA and nucleotide biosynthesis, which represent a novel and promising class of biofilm inhibitors. Finally, we discuss the activation of biofilm dispersal as a novel mode of action for anti-biofilm compounds.     

Quantifying biofilm structure using image analysis

We have developed and implemented methods of extracting morphological features from images of biofilms in order to quantify the characteristics of the inherent heterogeneity. This is a first step towards quantifying the relationship between biofilm heterogeneity and the underlying processes, such as mass-transport dynamics, substrate concentrations, and species variations. We have examined two categories of features, areal, which quantify the relative magnitude of the heterogeneity and textural, which quantify the microscale structure of the heterogeneous elements. The feature set is not exhaustive and has been restricted to two-dimensional images to this point. Included in this paper are the methods used to extract the structural information and the algorithms used to quantify the data. The features discussed are porosity, fractal dimension, diffusional length, angular second moment, inverse difference moment and textural entropy. We have found that some features are better predictors of biofilm behavior than others and we discuss possible future directions for research in this area.     

Assessing evolutionary relationships among microbes from whole-genome analysis

The determination and analysis of complete genome sequences have recently enabled many major advances to be made in the area of microbial evolutionary biology. These include the determination of the first genome of a Crenarchaeota, the suggestion that horizontal gene transfer may be the rule rather than the exception, and revelations about how genomes evolve on short timescales.     

细菌生物被膜检测与分析方法

细菌生物被膜(Biofilm,BF)由物体表面集聚生长的微生物群落和自身分泌的胞外物质构成,是造成细菌产生多重耐药性的原因之一。可靠、简单和快速的BF检测方法有助于有效预防和治疗相关疾病。基于不同原理的检测与分析方法已广泛用于BF研究中,本文从生物学方法、物理方法和化学方法等方面对BF检测方法进行总结,重点阐述显微镜技术在BF检测中的应用。并介绍了近年来发展的拉曼光谱、质谱成像、MALDI-TOF-MS等新技术,同时比较其优点和局限性,以便研究者找到最合适和最新的研究方法。     

生物膜定量分析仪对不同细菌早期生物膜形成能力的比较研究

目的通过生物膜定量分析仪来观察铜绿假单胞菌(Pseudomonas aeruginosa PAO1),变形链球菌(Streptococcus mutans UA159)以及大肠埃希菌(Escherichia coli MG1655)生物膜形成能力的不同,并以各菌株的吸光度值A600为参考,对3种菌株早期生物膜形成能力进行比较。方法通过向生物膜培养悬液中加入与细菌直径相近的磁性小珠,利用这些小珠在磁场中受到生物膜的位移约束力的原理,采用生物膜定量分析仪,定量比较3种菌株在生物膜形成上的差别。结果实验发现铜绿假单胞菌PAO1和大肠埃希菌MG1655的细菌增长速度基本相同,但铜绿假单胞菌PAO1的生物膜形成明显快于大肠埃希菌MG1655。大肠埃希菌MG1655和变形链球菌UA159的生物膜形成速度基本相同,但大肠埃希菌MG1655的细菌增长速度明显高于变形链球菌UA159。结论不同细菌有各自的生物膜形成模式。生物膜定量分析仪作为一种高效简便的检测手段,可用于生物膜早期形成的动态分析。     

Differential proteomic analysis of a polymicrobial biofilm

Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia exist in a polymicrobial biofilm associated with chronic periodontitis. The aim of this study was to culture these three species as a polymicrobial biofilm and to determine proteins important for bacterial interactions. In a flow cell all three species attached and grew as a biofilm; however, after 90 h of culture P. gingivalis and T. denticola were closely associated and dominated the polymicrobial biofilm. For comparison, planktonic cultures of P. gingivalis and T. denticola were grown separately in continuous culture. Whole cell lysates were subjected to SDS-PAGE, followed by in-gel proteolytic H(2)(16)O/H(2)(18)O labeling. From two replicates, 135 and 174 P. gingivalis proteins and 134 and 194 T. denticola proteins were quantified by LC-MALDI TOF/TOF MS. The results suggest a change of strategy in iron acquisition by P. gingivalis due to large increases in the abundance of HusA and HusB in the polymicrobial biofilm while HmuY and other iron/haem transport systems decreased. Significant changes in the abundance of peptidases and enzymes involved in glutamate and glycine catabolism suggest syntrophy. These data indicate an intimate association between P. gingivalis and T. denticola in a biofilm that may play a role in disease pathogenesis.