'Cells' and 'organisms' as a habitat for DNA.

Although the bulk of the hereditary information in bacteria is organized as a single chromosome, it has been known for some years now that bacteria may also carry pieces of self-replicating extrachromosomal DNA. These units are known as plasmids. Sometimes such plasmids carry the information necessary to give rise to mature bacterial viruses under appropriate conditions, but in other cases they specify the production of enzymes and other proteins which alter the bacterial phenotype. Plasmids are often inessential for survival of bacteria, although they may widen the range of environmental conditions under which they flourish. Thus plasmids may be thought of as adventitious additions to the genetic content of bacterial cells. Recently it has become clear that furthur organizational units of DNA are to be found in bacterial cells. These units are called insertion sequences and transposons. Unlike plasmids and the chromosome, however, these DNA units do not carry enough genetic information to specify their own independent replication: they must rely on plasmids or the chromosome for that purpose. Nevertheless they behave in many respects as independent functional units. Although it is possible to think of the chromosome, plasmids and transposons/insertion sequences as three distinct hierarchies of bacterial DNA, genes may move from one hierarchy to another; and such transitions have important implications for the evolution of bacterial populations. Moreover, their study in bacteria may throw much light on the type of DNA interactions occurring in higher cells.     

Modes of cytometric bacterial DNA pattern: a tool for pursuing growth

Analyses of DNA pattern provide an excellent tool to determine activity states of bacteria. Bacterial cell cycle behaviour is generally different from the eukaryotic one and is pre-determined by the bacteria's diversity within the phylogenetic tree, and their metabolic traits. As a result, every species creates its specific proliferation pattern that differs from every other one. Up to now, just few bacterial species have been investigated and little information is available concerning DNA cycling even in already known species. This prevents understanding of the complexity and diversity of ongoing bacterial interactions in many ecosystems or in biotechnology. Flow cytometry is the only possible technique to shed light on the dynamics of bacterial communities and DNA patterns will help to unlock the hidden principles of their life. This review provides basic knowledge about the molecular background of bacterial cell cycling, discusses modes of cell cycle phases and presents techniques to both obtain DNA patterns and to combine the contained information with physiological cell states.     

Whole genome plasticity in pathogenic bacteria

The exploitation of bacterial genome sequences has so far provided a wealth of new general information about the genetic diversity of bacteria, such as that of many pathogens. Comparative genomics uncovered many genome variations in closely related bacteria and revealed basic principles involved in bacterial diversification, improving our knowledge of the evolution of bacterial pathogens. A correlation between metabolic versatility and genome size has become evident. The degenerated life styles of obligate intracellular pathogens correlate with significantly reduced genome sizes, a phenomenon that has been termed "evolution by reduction". These mechanisms can permanently alter bacterial genotypes and result in adaptation to their environment by genome optimization. In this review, we summarize the recent results of genome-wide approaches to studying the genetic diversity of pathogenic bacteria that indicate that the acquisition of DNA and the loss of genetic information are two important mechanisms that contribute to strain-specific differences in genome content.     

Manipulation of autophagy by bacteria for their own benefit

Autophagy is the host innate immune system's first line of defense against microbial intruders. When the innate defense system recognizes invading bacterial pathogens and their infection processes, autophagic proteins act as cytosolic sensors that allow the autophagic pathway to be rapidly activated. However, many intracellular bacterial pathogens deploy highly evolved mechanisms to evade autophagic recognition, manipulate the autophagic pathway, and remodel the autophagosomal compartment for their own benefit. Here current topics regarding the recognition of invasive bacteria by the cytosolic innate immune system are highlighted, including autophagy and the mechanisms that enable bacteria to evade autophagy. Also highlighted are some selective examples of bacterial activities that manipulate the autophagic pathways for their own benefit.     

Phage as agents of lateral gene transfer

When establishing lysogeny, temperate phages integrate their genome as a prophage into the bacterial chromosome. Prophages thus constitute in many bacteria a substantial part of laterally acquired DNA. Some prophages contribute lysogenic conversion genes that are of selective advantage to the bacterial host. Occasionally, phages are also involved in the lateral transfer of other mobile DNA elements or bacterial DNA. Recent advances in the field of genomics have revealed a major impact by phages on bacterial chromosome evolution.     

Use of complex DNA and antibody microarrays as tools in functional analyses

While the deciphering of basic sequence information on a genomic scale is yielding complete genomic sequences in ever-shorter intervals, experimental procedures for elucidating the cellular effects and consequences of the DNA-encoded information become critical for further analyses. In recent years, DNA microarray technology has emerged as a prime candidate for the performance of many such functional assays. Technically, array technology has come a long way since its conception some 15 years ago, initially designed as a means for large-scale mapping and sequencing.The basic arrangement, however, could be adapted readily to serve eventually as an analytical tool in a large variety of applications. On their own or in combination with other methods, microarrays open up many new avenues of functional analysis.     

细菌自然转化的分子机制及生物学功能研究进展

基因的水平转移在细菌的进化中起着非常重要的作用。自然界中的细菌之间主要通过3种机制进行基因水平转移:由噬菌体介导的转导、接合转移和自然转化。自然转化是指自然感受态的细菌能够自发地从外界环境中摄取DNA分子并整合到自身基因组上的过程。该现象首先发现于肺炎链球菌,目前至少有83种细菌被发现具有发生自然转化的能力,其中革兰氏阳性菌以肺炎链球菌(Streptococcus pneumoniae,S. pneumoniae)为代表,革兰氏阴性菌以奈瑟氏菌(Neisseria)为代表,对其自然转化机制的研究和认识较为清楚,但不同细菌之间自然转化的机制有所差异。自然转化的生物学功能一直以来有以下几种推测:获取营养、修复DNA损伤、生物进化,而近年来对此认识争论不休。本文将详细描述细菌自然转化的分子机制,并对其主要的生物学功能争论焦点进行评述,以期对细菌自然转化有更深入的理解和认识。     

细菌c-di-AMP检测方法的研究进展

环二腺苷酸(cyclic diadenylate monophosphate,c-di-AMP)是一种广泛存在于细菌中的重要核苷酸第二信使分子,在病原体细菌,尤其是许多革兰阴性细菌中发挥着重要作用.研究表明,c-di-AMP在细菌生长、耐药性、抗应激、侵袭力和生物膜形成等方面担当不可取代的角色,同时参与激活和调节宿主的...     

Influence of Commonly Used Primer Systems on Automated Ribosomal Intergenic Spacer Analysis of Bacterial Communities in Environmental Samples

Due to the high diversity of bacteria in many ecosystems, their slow generation times, specific but mostly unknown nutrient requirements and syntrophic interactions, isolation based approaches in microbial ecology mostly fail to describe microbial community structure. Thus, cultivation independent techniques, which rely on directly extracted nucleic acids from the environment, are a well-used alternative. For example, bacterial automated ribosomal intergenic spacer analysis (B-ARISA) is one of the widely used methods for fingerprinting bacterial communities after PCR-based amplification of selected regions of the operon coding for rRNA genes using community DNA. However, B-ARISA alone does not provide any taxonomic information and the results may be severely biased in relation to the primer set selection. Furthermore, amplified DNA stemming from mitochondrial or chloroplast templates might strongly bias the obtained fingerprints. In this study, we determined the applicability of three different B-ARISA primer sets to the study of bacterial communities. The results from in silico analysis harnessing publicly available sequence databases showed that all three primer sets tested are specific to bacteria but only two primers sets assure high bacterial taxa coverage (1406f/23Sr and ITSF/ITSReub). Considering the study of bacteria in a plant interface, the primer set ITSF/ITSReub was found to amplify (in silico) sequences of some important crop species such as Sorghum bicolor and Zea mays. Bacterial genera and plant species potentially amplified by different primer sets are given. These data were confirmed when DNA extracted from soil and plant samples were analyzed. The presented information could be useful when interpreting existing B-ARISA results and planning B-ARISA experiments, especially when plant DNA can be expected.     

One by one: Single molecule tools for genomics.

Much of the effort in any genomics programme arises from the need to generate and purify large numbers of identical molecules, since most analytical tools rely on the analysis of bulk DNA. Biological steps such as bacterial cloning--commonly used to prepare bulk samples of defined DNA fragments--are capricious and introduce their own restrictions and distortions. The analysis of single molecules, either directly or by in vitro enzymatic amplification, makes possible the examination of native genomic DNA without the complications and restrictions of biological propagation. Techniques already exist for the in vitro propagation of genomic fragments and for genome mapping, and offer the advantages of speed, flexibility and predictable behaviour. Single molecule sequencing, for which many approaches are being developed, is more challenging, but offers even greater rewards in terms of throughput and read length.     

An RNA biology perspective on species-specific programmable RNA antibiotics

Our body is colonized by a vast array of bacteria the sum of which forms our microbiota. The gut alone harbors >1,000 bacterial species. An understanding of their individual or synergistic contributions to human health and disease demands means to interfere with their functions on the species level. Most of the currently available antibiotics are broad-spectrum, thus too unspecific for a selective depletion of a single species of interest from the microbiota. Programmable RNA antibiotics in the form of short antisense oligonucleotides (ASOs) promise to achieve precision manipulation of bacterial communities. These ASOs are coupled to small peptides that carry them inside the bacteria to silence mRNAs of essential genes, for example, to target antibiotic-resistant pathogens as an alternative to standard antibiotics. There is already proof-of-principle with diverse bacteria, but many open questions remain with respect to true species specificity, potential off-targeting, choice of peptides for delivery, bacterial resistance mechanisms and the host response. While there is unlikely a one-fits-all solution for all microbiome species, I will discuss how recent progress in bacterial RNA biology may help to accelerate the development of programmable RNA antibiotics for microbiome editing and other applications.     

Bacterial origin and community composition in the barley phytosphere as a function of habitat and presowing conditions

An understanding of the factors influencing colonization of the rhizosphere is essential for improved establishment of biocontrol agents. The aim of this study was to determine the origin and composition of bacterial communities in the developing barley (Hordeum vulgare) phytosphere, using denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes amplified from extracted DNA. Discrete community compositions were identified in the endorhizosphere, rhizoplane, and rhizosphere soil of plants grown in an agricultural soil for up to 36 days. Cluster analysis revealed that DGGE profiles of the rhizoplane more closely resembled those in the soil than the profiles found in the root tissue or on the seed, suggesting that rhizoplane bacteria primarily originated from the surrounding soil. No change in bacterial community composition was observed in relation to plant age. Pregermination of the seeds for up to 6 days improved the survival of seed-associated bacteria on roots grown in soil, but only in the upper, nongrowing part of the rhizoplane. The potential occurrence of skewed PCR amplification was examined, and only minor cases of PCR bias for mixtures of two different DNA samples were observed, even when one of the samples contained plant DNA. The results demonstrate the application of culture-independent, molecular techniques in assessment of rhizosphere bacterial populations and the importance of the indigenous soil population in colonization of the rhizosphere.     

Recruitment of TBK1 to cytosol‐invading Salmonella induces WIPI2‐dependent antibacterial autophagy

Mammalian cells deploy autophagy to defend their cytosol against bacterial invaders. Anti‐bacterial autophagy relies on the core autophagy machinery, cargo receptors, and “eat‐me” signals such as galectin‐8 and ubiquitin that label bacteria as autophagy cargo. Anti‐bacterial autophagy also requires the kinase TBK1, whose role in autophagy has remained enigmatic. Here we show that recruitment of WIPI2, itself essential for anti‐bacterial autophagy, is dependent on the localization of catalytically active TBK1 to the vicinity of cytosolic bacteria. Experimental manipulation of TBK1 recruitment revealed that engagement of TBK1 with any of a variety of Salmonella‐associated “eat‐me” signals, including host‐derived glycans and K48‐ and K63‐linked ubiquitin chains, suffices to restrict bacterial proliferation. Promiscuity in recruiting TBK1 via independent signals may buffer TBK1 functionality from potential bacterial antagonism and thus be of evolutionary advantage to the host.     

Enterococcal biofilm—A nidus for antibiotic resistance transfer?

Enterococci, which are on the WHO list of priority pathogens, are commonly encountered in hospital acquired infection and are becoming increasing significant due to the development of strains resistant to multiple antibiotics. Enterococci are also important microorganisms in the environment, and their presence is frequently used as an indicator of faecal pollution. Their success is related to their ability to survive within a broad range of habitats and the ease by which they acquire mobile genetic elements, including plasmids, from other bacteria. The enterococci are frequently present within a bacterial biofilm, which provides stability and protection to the bacterial population along with an opportunity for a variety of bacterial interactions. Enterococci can accept extrachromosomal DNA both from within its own species and from other bacterial species, and this is enhanced by the proximity of the donor and recipient strains. It is this exchange of genetic material that makes the role of biofilms such an important aspect of the success of enterococci. There remain many questions regarding the most suitable model systems to study enterococci in biofilms and regarding the transfer of genetic material including antibiotic resistance in these biofilms. This review focuses on some important aspects of biofilm in the context of horizontal gene transfer (HGT) in enterococci.     

革兰氏阴性菌外膜囊泡作为亚单位疫苗的研究进展北大核心CSCD

外膜囊泡(OMVs,Outer membrane vesicles)是一种在革兰氏阴性菌甚至某些革兰氏阳性菌中普遍存在的包含生物学活性物质的囊泡状结构,其大小在20–250 nm之间。其组成成分包括脂多糖、外膜蛋白、磷脂、DNA以及在形成过程中被外膜包裹的周质成分等。由于外膜囊泡不能复制且含有大量的细菌抗原,并能有效激活免疫系统,所以被认为是极具潜力的疫苗候选。虽然外膜囊泡从发现至今有50多年的历史,但针对其作为疫苗的潜力探究最近几年才开始,中国关于这方面的文献报道还很少。本文从外膜囊泡诱导免疫应答的机制以及其作为疫苗的研究进展两个方面概述了外膜囊泡可以作为一种新颖的防控疾病的疫苗策略,为今后外膜囊泡疫苗的深入研究提供参考。     

The food safety perspective of antibiotic resistance

Bacterial antimicrobial resistance in both the medical and agricultural fields has become a serious problem worldwide. Antibiotic resistant strains of bacteria are an increasing threat to animal and human health, with resistance mechanisms having been identified and described for all known antimicrobials currently available for clinical use. There is currently increased public and scientific interest regarding the administration of therapeutic and sub-therapeutic antimicrobials to animals, due primarily to the emergence and dissemination of multiple antibiotic resistant zoonotic bacterial pathogens. This issue has been the subject of heated debates for many years, however, there is still no complete consensus on the significance of antimicrobial use in animals, or resistance in bacterial isolates from animals, on the development and dissemination of antibiotic resistance among human bacterial pathogens. In fact, the debate regarding antimicrobial use in animals and subsequent human health implications has been going on for over 30 years, beginning with the release of the Swann report in the United Kingdom. The latest report released by the National Research Council (1998) confirmed that there were substantial information gaps that contribute to the difficulty of assessing potential detrimental effects of antimicrobials in food animals on human health. Regardless of the controversy, bacterial pathogens of animal and human origin are becoming increasingly resistant to most frontline antimicrobials, including expanded-spectrum cephalosporins, aminoglycosides, and even fluoroquinolones. The lion's share of these antimicrobial resistant phenotypes is gained from extra-chromosomal genes that may impart resistance to an entire antimicrobial class. In recent years, a number of these resistance genes have been associated with large, transferable, extra-chromosomal DNA elements, called plasmids, on which may be other DNA mobile elements, such as transposons and integrons. These DNA mobile elements have been shown to transmit genetic determinants for several different antimicrobial resistance mechanisms and may account for the rapid dissemination of resistance genes among different bacteria. The increasing incidence of antimicrobial resistant bacterial pathogens has severe implications for the future treatment and prevention of infectious diseases in both animals and humans. Although much scientific information is available on this subject, many aspects of the development of antimicrobial resistance still remain uncertain. The emergence and dissemination of bacterial antimicrobial resistance is the result of numerous complex interactions among antimicrobials, microorganisms, and the surrounding environments. Although research has linked the use of antibiotics in agriculture to the emergence of antibiotic-resistant foodborne pathogens, debate still continues whether this role is significant enough to merit further regulation or restriction.     

Bacterial signals and antagonists: the interaction between bacteria and higher organisms

It is now well established that bacteria communicate through the secretion and uptake of small diffusable molecules. These chemical cues, or signals, are often used by bacteria to coordinate phenotypic expression and this mechanism of regulation presumably provides them with a competitive advantage in their natural environment. Examples of coordinated behaviors of marine bacteria which are regulated by signals include swarming and exoprotease production, which are important for niche colonisation or nutrient acquisition (e.g. protease breakdown of substrate). While the current focus on bacterial signalling centers on N-Acylated homoserine lactones, the quorum sensing signals of gram-negative bacteria, these are not the only types of signals used by bacteria. Indeed, there appears to be many other types of signals produced by bacteria and it also appears that a bacterium may use multiple classes of signals for phenotypic regulation. Recent work in the area of marine microbial ecology has led to the observation that some marine eukaryotes secrete their own signals which compete with the bacterial signals and thus inhibit the expression of bacterial signalling phenotypes. This type of molecular mimicry has been well characterised for the interaction of marine prokaryotes with the red alga, Delisea pulchra.     

Microbial genomics: rhetoric or reality?

The availability of complete genome sequences of many bacterial species is facilitating numerous computational approaches for understanding bacterial genomes. One of the major incentives behind the genome sequencing of many pathogenic bacteria is the desire to better understand their diversity and to develop new approaches for controlling human diseases caused by these microorganisms. This task has become even more urgent with the rapid evolution of antibiotic resistance among many bacterial pathogens. Novel drug targets are required in order to design new antimicrobials against antibiotic-resistant pathogens. The complete genome sequences of an ever increasing number of pathogenic microbes constitute an invaluable resource and provide lead information on potential drug targets. This review focuses on in silico analyses of microbial genomes, their host-specific adaptations, with specific reference to genome architecture, design, evolution, and trends in computational identification of microbial drug targets. These trends underscore the utility of genomic data for systematic in silico drug target identification in the post-genomic era.