The Continuous Culture of Luminous Bacteria: a Luminostat

A method has been designed for the continuous culture of luminous bacteria. The control system for the culture uses a combination of luminescence and optical density as a light signal received by a photomultiplier. This combined signal operates pumps which exchange the growth medium. Using this method, a culture of brightly luminescing bacteria was maintained for periods up to 3 weeks.     

Measurement of Horizontal and Vertical Movement of Ralstonia solanacearum in Soil

Two model systems were constructed to measure horizontal and vertical movement of bacteria in soil. These systems were applied to measuring movement of Ralstonia solanacearum (race 1, biovar 3), a causal agent of bacterial wilt of tomato, in andosol and sand at 28°C. The first system was used to measure horizontal movement of the bacteria in soil packed in a narrow horizontal frame. Suspension of the pathogen was applied to soil at one end of the frame, and bacterial number per gram of soil was measured over distance from the inoculation point after 4 days. Horizontal movement of R. solanacearum in supersaturated soil, but without flow, was possibly due to diffusion and the front advanced at 2.2 cm/day in andosol, and at 8.1 cm/day in sand. Using the same experimental system, but applying water inflow to one end of the frame only, the bacterium was detected at the front of water in andosol and sand. The front of the distribution advanced at 20.4 cm/h in andosol and 66.3 cm/h in sand. In the second experimental system, a cylinder of soil packed in a short tube was soaked with water, and soil at the top of the tube was inoculated with bacterial suspension. Immediately, soil cylinders were turned upward, and the bacterial number per gram of soil was measured along vertical distance from the inoculation point after 7 days. Using the system with andosol, the capillary water front rose to 32.5 cm over 7 days after inoculation, and R. solanacearum reached to 18.8 cm height. In sand, capillary water rose to 20.0 cm and the bacteria reached to 16.3 cm height.     

Complement-mediated bactericidal activity of human milk to a serum-susceptible strain of E. coli 0111

There has been a lot of controversy concerning the physiological significance of the complement system in human breast-milk. This is mainly due to the observation that human milk contains predominantly non-inflammatory and many anti-inflammatory factors, while simultaneously protecting the infant against a wide range of infectious and other diseases. The present study was carried out to assess the contribution of the complement system to the bactericidal activity of the human colostrum and early lactational milk. Using a serum-sensitive strain of Escherichia coli, different fractions of human breast-milk were assessed for their ability to kill the bacteria, with and without inactivation of their complement components, in comparison to another strain of the bacteria species. Deposition of activated C3 fragments on the killed bacteria, using an established ELISA technique, was demonstrated, further proving that the human milk complement could be activated in vitro. The bactericidal activities of human milk were almost completely abolished by complement heat inactivation at 56 degrees C or by the addition of EDTA.     

Measurement of Predation and Biofilm Formation under Different Ambient Oxygen Conditions Using a Simple Gasbag-Based System

Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus are Gram-negative bacteria characterized by predatory behavior. The aim of this study was to evaluate the ability of the predators to prey in different oxygen environments. When placed on an orbital shaker, a positive association between the rate of aeration and predation was observed. To further examine the effects of elevated ambient oxygen levels on predation, a simple gasbag system was developed. Using the system, we were able to conduct experiments at ambient oxygen levels of 3% to 86%. When placed in gasbags and inflated with air, 50% O₂, and 100% O₂, positive predation was seen on both planktonic and biofilm-grown prey cells. However, in low-oxygen environments, predatory bacteria were able to attack only prey cells grown as biofilms. To further evaluate the gasbag system, biofilm development of Gram-positive and Gram-negative microorganisms was also measured. Although the gasbag system was found to be suitable for culturing bacteria that require a low-oxygen environment, it was not capable of supporting, with its current configuration, the growth of obligate anaerobes in liquid or agar medium.     

Plasmid transformation of Ruminococcus albus by means of high-voltage electroporation

To apply recombinant DNA techniques to the genetic manipulation of cellulolytic ruminal bacteria, a plasmid vector transformation system must be available. The objective of this work was to develop a system for plasmid transformation of Ruminococcus albus. Using high voltage electrotransformation, pSC22 and pCK17 plasmid vectors, derived from lactic acid bacteria plasmids and replicating via single-stranded DNA intermediate, were successfully introduced into three freshly isolated R. albus strains and into R. albus type strain ATCC 27210. The optimization of the electrotransformation condition raised the electroporation efficiency up to 3 x 10(5) transformants per microgram of pSC22 plasmid.     

Screening for lipase-producing microorganisms with a continuous cultivation system

Summary The enrichment of lipase-producing bacteria, isolated from the environment, was evaluated using a continuous cultivation system. Continuous cultivations were performed using a synthetic medium where the carbon and energy source, triolein, was provided physically entrapped in an external loop. Using this system, pure cultivations of Pseudomonas fluorescens 378 showed an increase in lipase-producing ability, instead of declining as in more conventional systems. Enrichment cultures were obtained with environmental samples originating from a vegetable oil processing plant using the same system. Three types of lipase-producing bacteria were identified: P. alcaligenes, Enterobacter intermedium and a Gram-negative, oxidase-positive rod. Offprint requests to: E. Lie     

Rapid detection and quantification of five periodontopathic bacteria by real-time PCR

The quantity of periodontopathic bacteria in plaque samples is an important determinant for understanding the etiologic role of bacteria. The real-time PCR method was used to detect and quantify periodontopathic bacteria, such as Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Porphyromonas gingivalis, Treponema denticola, and Treponema socranskii, in saliva and subgingival plaque samples. There was good agreement between the results of conventional PCR and real-time PCR methods. Using the LightCycler system we were able to determine the amount of periodontopathic bacteria within an hour. The real-time PCR method was linear for samples containing from 10(3) to more than 10(8) cells (r2 = 0.999). The application of the real-time PCR method should be useful in the rapid detection and quantification of periodontopathic bacteria in clinical samples.     

Antagonistic interactions among marine bacteria impede the proliferation of Vibrio cholerae

Changes in global climate have raised concerns about the emergence and resurgence of infectious diseases. Vibrio cholerae is a reemerging pathogen that proliferates and is transported on marine particles. Patterns of cholera outbreaks correlate with sea surface temperature increases, but the underlying mechanisms for rapid proliferation of V. cholerae during ocean warming events have yet to be fully elucidated. In this study, we tested the hypothesis that autochthonous marine bacteria impede the spread of V. cholerae in the marine environment. It was found that some marine bacteria are capable of inhibiting the growth of V. cholerae on surfaces and that bacterial isolates derived from pelagic particles show a greater frequency of V. cholerae inhibition than free-living bacteria. Vibrio cholerae was less susceptible to antagonism at higher temperatures, such as those measured during El Ni?o-Southern Oscilliation and monsoonal events. Using a model system employing green fluorescent protein-labeled bacteria, we found that marine bacteria can directly inhibit V. cholerae colonization of particles. The mechanism of inhibition in our model system was linked to the biosynthesis of andrimid, an antibacterial agent. Antibiotic production by the model antagonistic strain decreased at higher temperatures, thereby explaining the increased competitiveness of V. cholerae under warmer conditions. These findings suggest that bacterium-bacterium antagonism is a contributing mechanism in regulating the proliferation of V. cholerae on marine particles.     

The twin arginine translocation system contributes to symbiotic colonization of Euprymna scolopes by Vibrio fischeri

In many bacteria, the twin arginine translocation (Tat) system transports folded proteins across the cytoplasmic membrane, and these proteins can play a role in symbiotic or pathogenic infections. A role for the Vibrio fischeri Tat system was identified during symbiotic colonization of its host Euprymna scolopes, demonstrating a function for the Tat system in host colonization by a member of the Vibrionaceae. Using bioinformatics, mutant analyses, and green fluorescent protein fusions, a set of Tat-targeted proteins in V. fischeri was identified.     

实验动物屏障系统微生物动态状况

目的研究运行中的实验动物屏障系统微生物的情况。方法采用沉降菌法、棉拭子法等方法,研究运行中的屏障系统不同区域、不同环境指标下屏障系统内微生物的状况。结果动态下的屏障系统微生物情况与国标GB14925-2001中静态环境有较大不同,动物饲养室和动物实验室沉降菌浓度远高于静态要求;辅助区域在规范化消毒及严格管理的情况下,能达到国标要求。屏障系统的微生物情况存在一定的昼夜变化规律,在晚间出现峰值。结论合适的换气次数可有效控制实验动物屏障系统的沉降菌浓度;加强消毒及硬件的管理,是屏障系统内环境稳定的保障。     

A UV‐C LED‐based unit for continuous decontamination of the sheath fluid in a flow‐cytometric cell sorter

Aseptic cell sorting is challenging, especially when a flow‐cytometric cell sorter is not operated in a sterile environment. The sheath fluid system of a cell sorter may be contaminated with germs such as bacteria, yeasts, viruses, or fungi. Thus, a regular chemical cleaning procedure is required to prepare a sorter for aseptic cell sorting by flushing the fluidic system. However, this procedure is time consuming, and most importantly, the researcher can never be sure that the cleaning process was successful. Here we present a method in which the sheath fluid of a cell sorter was decontaminated by irradiation with UV‐C light using a flow‐through principle. Using this principle, we were able to achieve a 5 log reduction of bacteria in the sheath fluid.     

用pKKH质粒在大肠杆菌中表达HuIFN－β

应用ＤＮＡ重组技术,将ＨｕＩＦＮ－β基因插入到质粒ｐＫＫＨ的ｔａｃ启动子下游,转化大肠杆菌ＪＭ１０１和ＪＭ１０３,经ＩＰＴＧ诱导,表达ＨｕＩＦＮ－β,收集并裂解细菌,用Ｗｉｓｈ－ＶＳＶ系统细胞病变抑制法检测生物学活性为２．１８×１０８－８．７×１０８ＩＵ／Ｌ菌液。经初步纯化ＳＤＳ－ＰＡＧＥ电泳可见分子量为２０ＫＤ较纯的表达带。     

SPEX, a System for the Expression of Recombinant Proteins from Gram-Positive Bacterial Vectors

Using a conserved pathway for surface protein extrusion, a system has been developed for the expression and secretion of proteins from gram-positive bacteria. As proof-of-concept, theStreptococcus gordoniiChallis strain has been engineered to express a series of recombinant proteins fused to the conserved region of the M6 protein ofStreptococcus pyogenes.In the prototype surface protein expression system, the recombinant M6 protein is anchored to the surface ofS. gordoniicells expressing it. In order to overexpress the protein and easily purify it away from the bacteria, the protein was modified to enable it to be secreted into the medium. To accomplish this, a stop codon was introduced into the gene just prior to the anchor region using site-directed mutagenesis. Using enzyme-linked immunosorbent assays, it was possible to quantitate the amount of protein expressed using this system. With little or no optimization, 3 mg of protein per liter of culture was expressed and secreted into the medium of a bacterial culture grown to an OD₆₀₀equal to 1.0. This system should be broadly applicable for the expression and secretion of a variety of proteins (antigens, hormones, and enzymes) directly into the medium.     

Recognition of gram-positive intestinal bacteria by hybridoma- and colostrum-derived secretory immunoglobulin A is mediated by carbohydrates

Humans live in symbiosis with 10(14) commensal bacteria among which >99% resides in their gastrointestinal tract. The molecular bases pertaining to the interaction between mucosal secretory IgA (SIgA) and bacteria residing in the intestine are not known. Previous studies have demonstrated that commensals are naturally coated by SIgA in the gut lumen. Thus, understanding how natural SIgA interacts with commensal bacteria can provide new clues on its multiple functions at mucosal surfaces. Using fluorescently labeled, nonspecific SIgA or secretory component (SC), we visualized by confocal microscopy the interaction with various commensal bacteria, including Lactobacillus, Bifidobacteria, Escherichia coli, and Bacteroides strains. These experiments revealed that the interaction between SIgA and commensal bacteria involves Fab- and Fc-independent structural motifs, featuring SC as a crucial partner. Removal of glycans present on free SC or bound in SIgA resulted in a drastic drop in the interaction with gram-positive bacteria, indicating the essential role of carbohydrates in the process. In contrast, poor binding of gram-positive bacteria by control IgG was observed. The interaction with gram-negative bacteria was preserved whatever the molecular form of protein partner used, suggesting the involvement of different binding motifs. Purified SIgA and SC from either mouse hybridoma cells or human colostrum exhibited identical patterns of recognition for gram-positive bacteria, emphasizing conserved plasticity between species. Thus, sugar-mediated binding of commensals by SIgA highlights the currently underappreciated role of glycans in mediating the interaction between a highly diverse microbiota and the mucosal immune system.     

Purification and properties of the dnaJ replication protein of Escherichia coli

The Escherichia coli dnaJ gene was originally discovered because mutations in it blocked bacteriophage lambda DNA replication. Some of these mutations were subsequently shown to interfere with bacterial growth at high temperature, suggesting that dnaJ is an essential protein for the host as well. The first step in purifying the dnaJ protein was to overproduce it at least 50-fold by subcloning its gene into the pMOB45 runaway plasmid. The second step was the development of an in vitro system to assay for its activity. A Fraction II extract from dnaJ259 mutant bacteria was shown to be unable to replicate lambda dv DNA unless supplemented with an exogenous source of wild-type dnaJ protein. Using this complementation assay we purified the dnaJ protein to homogeneity from the membrane fraction of an overproducing strain of bacteria. The purified dnaJ protein was shown to be a basic (pI 8.5), yet hydrophobic, protein of Mr 37,000 and 76,000 under denaturing and native conditions, respectively, and to exhibit affinity for both single- and double-stranded DNA. Using a partially purified lambda dv replication system dependent on the presence of the lambda O and P initiator proteins and at least the host dnaB, dnaG, dnaJ, dnaK, single-stranded DNA-binding protein, gyrase, RNA polymerase holoenzyme, and DNA polymerase III holoenzyme, we have shown that the dnaJ protein is required at a very early step in the DNA replication process.     

The Isolation and Characterisation of a Carbocyclic Epoxide-Degrading Corynebacterium sp

A two-phase aqueous/organic isolation system was developed for the isolation of epoxide-degrading bacteria. The potential of this system, for the isolation of cyclohexene oxide-degrading bacteria, was assessed by comparison to an analogous system lacking co-solvent. Using the biphasic isolation strategy, an epoxide-degrading Corynebacterium sp. designated C12, was isolated and was shown to grow on cyclohexene oxide as sole source of carbon and energy. Epoxide degradation appeared to proceed via a diol intermediate implicating the involvement of an epoxide hydrolase. The epoxide hydrolase of Corynebacterium sp. C12 was shown to have activity towards a range of terminal, sub-terminal and cyclic substrates. The enantioselectivity of the hydrolysis reaction was largely dependent on the nature of the substrate. In a series of biotransformations allowed to proceed to 50% substrate conversion, the remaining epoxide ranged from low (5% ee) to moderate (60% ee) optical purity.     

Mathematical modeling of bacterial virulence and host-pathogen interactions in the Dictyostelium/Pseudomonas system

We present some studies on the mechanisms of pathogenesis based on experimental work and on its interpretation through a mathematical model. Using a collection of clinical strains of the opportunistic human pathogen Pseudomonas aeruginosa, we performed co-culture experiments with Dictyostelium amoebae, to investigate the two organisms’ interaction, characterized by a cross action between amoeba, feeding on bacteria, and bacteria exerting their pathogenic action against amoeba. In order to classify bacteria virulence, independently of this cross interaction, we have also performed killing experiments of bacteria against the nematode Caenorhabditis elegans.A mathematical model was developed to infer how the populations of the amoeba-bacteria system evolve according to a number of parameters, taking into account the specific features underlying the interaction. The model does not fall within the class of traditional prey-predator models because not only does an amoeba feed on bacteria, but also it is in turn attacked by them; thus the model must include a feedback term modeling this further interaction aspect. The model shows the existence of multiple steady states and the resulting behavior of the solutions, showing bi-stability of the system, gives a qualitative explanation of the co-culture experiments.     

A method adapting microarray technology for signature-tagged mutagenesis of Desulfovibrio desulfuricans G20 and Shewanella oneidensis MR-1 in anaerobic sediment survival experiments

Signature-tagged mutagenesis (STM) is a powerful technique that can be used to identify genes expressed by bacteria during exposure to conditions in their natural environments. To date, there have been no reports of studies in which this approach was used to study organisms of environmental, rather than pathogenic, significance. We used a mini-Tn10 transposon-bearing plasmid, pBSL180, that efficiently and randomly mutagenized Desulfovibrio desulfuricans G20 in addition to Shewanella oneidensis MR-1. Using these organisms as model sediment-dwelling anaerobic bacteria, we developed a new screening system, modified from former STM procedures, to identify genes that are critical for sediment survival. The screening system uses microarray technology to visualize tags from input and output pools, allowing us to identify those lost during sediment incubations. While the majority of data on survival genes identified will be presented in future papers, we report here on chemotaxis-related genes identified by our STM method in both bacteria in order to validate our method. This system may be applicable to the study of numerous environmental bacteria, allowing us to identify functions and roles of survival genes in various habitats.     

Diversity in CRISPR‐based immunity protects susceptible genotypes by restricting phage spread and evolution

Diversity in host resistance often associates with reduced pathogen spread. This may result from ecological and evolutionary processes, likely with feedback between them. Theory and experiments on bacteria–phage interactions have shown that genetic diversity of the bacterial adaptive immune system can limit phage evolution to overcome resistance. Using the CRISPR–Cas bacterial immune system and lytic phage, we engineered a host–pathogen system where each bacterial host genotype could be infected by only one phage genotype. With this model system, we explored how CRISPR diversity impacts the spread of phage when they can overcome a resistance allele, how immune diversity affects the evolution of the phage to increase its host range and if there was feedback between these processes. We show that increasing CRISPR diversity benefits susceptible bacteria via a dilution effect, which limits the spread of the phage. We suggest that this ecological effect impacts the evolution of novel phage genotypes, which then feeds back into phage population dynamics.     

The alternative sigma factor HrpL negatively modulates the flagellar system in the phytopathogenic bacterium Erwinia amylovora under hrp-inducing conditions

In this work we present evidence of an opposite regulation in the phytopathogenic bacteria Erwinia amylovora between the virulence-associated Type III secretion system (TTSS) and the flagellar system. Using loss-of-function mutants we show that motility enhanced the virulence of wild-type bacteria relative to a nonmotile mutant when sprayed on apple seedlings with unwounded leaves. Then we demonstrated through analyses of motility, flagellin export and visualization of flagellar filament that HrpL, the positive key regulator of the TTSS, also down-regulates the flagellar system. Such a dual regulation mediated by an alternative sigma factor of the TTSS appears to be a level of regulation between virulence and motility not yet described among Proteobacteria.