Microbial genomics: tropical treasure?

A Brazilian consortium has unveiled the genomic DNA sequence of the purple-pigmented bacterium Chromobacterium violaceum, a dominant component of the tropical soil microbiota. The sequence provides insight into the abundant potential of this organism for biotechnological and pharmaceutical applications.     

紫色色杆菌感染临床特症及耐药谱分析

目的 研究紫色色杆菌感染的临床特征与病原体的生物学特性以及耐药分析,以提高临床对紫色色杆菌感染的诊断和治疗.方法 将患者的血液、浓液标本进行培养,分离并把分离获得的菌株在VITEK-32微生物鉴定仪中进行种的鉴定及药物敏感性试验,用小白鼠进行该病原体的毒力试验.结果 12种抗菌药物中舒普深、亚胺培南、特治星（派拉西林/他唑巴坦）、马斯平（头孢吡肟）及喹喏酮类抗菌药物对紫色色杆菌均有较强的抗菌作用,而氨苄西林、头孢唑啉、庆大霉素、妥布霉素、氨曲南、头孢曲松和头孢他啶等抗菌药物抗菌效果不佳.毒力试验结果表明该菌有较强毒力.结论 紫色色杆菌是一种革兰阴性杆菌,毒力强.可引起感染部位脓肿,并可侵入体内各脏器及血液引发脏器的脓肿,脓毒血症是该菌感染的临床特症,重者可引起各脏器功能衰退,中毒性休克甚至死亡.紫色色杆菌感染病情急,进展快,死亡率高,因此早发现,及早有效性治疗极为重要.     

Isolation of Chromobacterium spp. from foods, soil, and water.

Chromobacterium violaceum, a soil and water inhabitant, has been implicated in human disease with a high mortality rate, particularly in the southeastern United States. The psychrotrophic Chromobacterium lividum has been isolated from foods, water, and soil, but is not considered pathogenic. To determine the distribution of Chromobacterium spp. in soil, water, and foods in the Gainesville area, we evaluated Bennett, Ryalls and Moss, and Aeromonas membrane agars for their ability to recover these organisms from various samples when incubated at 25 or 35 degrees C. Bennett agar was best for the isolation of both species when incubated at 25 degrees C; however, at 35 degrees C, Aeromonas membrane agar gave the highest recoveries of C. violaceum. C. violaceum was recovered only from soil and water, whereas C. lividum was frequently recovered from foods as well as soil and water.     

The Serratia-type hemolysin of Chromobacterium violaceum

Chromobacterium violaceum is a Gram-negative opportunistic human pathogen and an inhabitant of tropical soils and waterways. Although known primarily for the synthesis of the pigment violacein, and more recently as a reporter strain for quorum sensing, clinical reports of chromobacteriosis comprise the largest block of published literature on this organism. Genome sequencing has revealed many potential virulence factors in this microorganism, and this paper establishes the presence in C. violaceum of a Serratia type-hemolysin (ChlA) and transporter (ChlB). We also show that the hemolysin operon includes a third gene (chlC) that is predicted to encode a phosphorylation domain similar to the receiver domain of response regulators in bacterial signal transduction systems.     

Identification of Chromobacterium violaceum genes with potential biotechnological application in environmental detoxification

Chromobacterium violaceum is a Gram-negative bacterium found in a wide variety of tropical and subtropical ecosystems. The complete genome sequence of C. violaceum ATCC 12472 is now available, and it has considerable biotechnological potential for various applications, such as environmental detoxification, as well as medical and agricultural use. We examined the biotechnological potential of C. violaceum for environmental detoxification. Three operons, comprising the ars operon, involved in arsenic resistance, the cyn operon, involved in cyanate detoxification, and the hcn operon, encoding a cyanase, responsible for biogenic production of cyanide, as well as an open reading frame, encoding an acid dehalogenase, were analyzed in detail. Probable catalytic mechanisms for the enzymes were determined, based on amino acid sequence comparisons and on published structural information for these types of proteins.     

Transport genes of Chromobacterium violaceum: an overview

The complete genome sequence of the free-living bacterium Chromobacterium violaceum has been determined by a consortium of laboratories in Brazil. Almost 500 open reading frames (ORFs) coding for transport-related membrane proteins were identified in C. violaceum, which represents 11% of all genes found. The main class of transporter proteins is the primary active transporters (212 ORFs), followed by electrochemical potential-driven transporters (154 ORFs) and channels/pores (62 ORFs). Other classes (61 ORFs) include group translocators, transport electron carriers, accessory factors, and incompletely characterized systems. Therefore, all major categories of transport-related membrane proteins currently recognized in the Transport Protein Database (http://tcdb.ucsd.edu/tcdb) are present in C. violaceum. The complex apparatus of transporters of C. violaceum is certainly an important factor that makes this bacterium a dominant microorganism in a variety of ecosystems in tropical and subtropical regions. From a biotechnological point of view, the most important finding is the transporters of heavy metals, which could lead to the exploitation of C. violaceum for bioremediation.     

Drug resistance in Chromobacterium violaceum

Chromobacterium violaceum is a free-living bacterium commonly found in aquatic habitats of tropical and subtropical regions of the world. This bacterium is able to produce a large variety of products of biotechnological and pharmacological use. Although C. violaceum is considered to be non-pathogenic, some cases of severe infections in humans and other animals have been reported. Genomic data on the type strain ATCC 12472(T) has provided a comprehensive basis for detailed studies of pathogenicity, virulence and drug resistance genes. A large number of open reading frames associated with various mechanisms of drug resistance were found, comprising a remarkable feature of this organism. Amongst these, beta-lactam (penicillin and cephalosporin) and multidrug resistance genes (drug efflux pumps) were the most numerous. In addition, genes associated with bacitracin, bicyclomycin, chloramphenicol, kasugamycin, and methylenomycin were also found. It is postulated that these genes contribute to the ability of C. violaceum to compete with other bacteria in the environment, and also may help to explain the common drug resistance phenotypes observed in infections caused by this bacterium.     

An analysis of type-III secretion gene clusters in Chromobacterium violaceum

Chromobacterium violaceum is an environmental Gram-negative bacterium that is common in soil and water in tropical and sub-tropical regions. It is also a model organism for studying quorum-sensing and is a rare but deadly human pathogen. Recent completion of the genome sequence of C. violaceum strain ATCC 12472 revealed the presence of genes associated with type-III secretion systems (TTSSs). One of these systems resembles the Spi-1 system found in Salmonella enterica, whereas another is similar to the Spi-2 system from the same organism. Here, we present a detailed analysis and a fresh annotation of the two gene clusters. Moreover, we highlight the presence of several genes encoding putative type-III effector proteins that lead us to predict that this organism can manipulate vesicular trafficking, the actin cytoskeleton and apoptotic pathways within mammalian cells to its own advantage.     

DNA repair in Chromobacterium violaceum

Chromobacterium violaceum is a Gram-negative beta-proteobacterium that inhabits a variety of ecosystems in tropical and subtropical regions, including the water and banks of the Negro River in the Brazilian Amazon. This bacterium has been the subject of extensive study over the last three decades, due to its biotechnological properties, including the characteristic violacein pigment, which has antimicrobial and anti-tumoral activities. C. violaceum promotes the solubilization of gold in a mercury-free process, and has been used in the synthesis of homopolyesters suitable for the production of biodegradable polymers. The complete genome sequence of this organism has been completed by the Brazilian National Genome Project Consortium. The aim of our group was to study the DNA repair genes in this organism, due to their importance in the maintenance of genomic integrity. We identified DNA repair genes involved in different pathways in C. violaceum through a similarity search against known sequences deposited in databases. The phylogenetic analyses were done using programs of the PHILYP package. This analysis revealed various metabolic pathways, including photoreactivation, base excision repair, nucleotide excision repair, mismatch repair, recombinational repair, and the SOS system. The similarity between the C. violaceum sequences and those of Neisserie miningitidis and Ralstonia solanacearum was greater than that between the C. violaceum and Escherichia coli sequences. The peculiarities found in the C. violaceum genome were the absence of LexA, some horizontal transfer events and a large number of repair genes involved with alkyl and oxidative DNA damage.     

具有群体感应抑制效果芽孢杆菌的筛选与鉴定

细菌性疾病是危害水产业健康发展的主要原因之一,从抑制调控致病菌毒力表达的群体感应（Quorum sensing,QS）入手是水产动物疾病防治研究新的热点方向。研究以紫色杆菌 （Chromobacterium violaceum）为报告菌株,采用T型划线和滤纸片法,对分离自异育银鲫肠道及实验室保存的芽孢杆菌F3-1、F3-2、F6-4、F6-5、X77、X93、X3914进行分析测测试,筛选具有群体感应抑制效果的菌株。T型划线结果显示菌株F3-1能够抑制紫色杆菌紫色菌素的产生;滤纸片法结果显示抑制紫色杆菌紫色菌素产生的主要物质存在于菌株F3-1的胞外产物中。试验提取了菌株F3-1的培养物的粗提物,测定粗提物浓度分别在0、50、100、200、300 mg/L时紫色杆菌菌素在585 nm处的光密度值,结果显示随着粗提物浓度的增加其色素的产量显著降低（P0.05）,当粗提物浓度超过200 mg/L时,试管中观察不到紫色菌素存在;利用硫酸铵盐析的方法,提取了菌株F3-1的粗提物中蛋白产物,滤纸片扩散法结果显示表明其胞外蛋白是紫色杆菌QS系统的抑制物质。通过分子生物学16S rDNA鉴定,确定菌株F3-1为短小芽孢杆菌（Bacillus pumilus,GenBank accession No. JX984444）。电镜照片显示该菌具有短杆状特征,因此,分离自异育银鲫肠道的短小芽孢杆菌F3-1具有抑制细菌群体感应的作用。       

In vitro testing for genotoxicity of violacein assessed by Comet and Micronucleus assays

Chromobacterium violaceum is a Gram (-) bacteria found in water samples and soils from tropical and subtropical regions of the world. Violacein, the major pigment produced by these bacteria, has been shown to have antibiotic, antitumoral and trypanocidal activities. In the present work, the genotoxicity of violacein was investigated in four different cell lines by using the alkaline Comet assay and in VERO cells using the Micronucleus test. In the alkaline Comet assay, violacein, when tested at concentrations ranging from 0.19 to 1.5 microM, did not induce a significant increase in DNA damage in HEp-2 and MA104 cells. However, violacein was positive for DNA damage in FRhK-4 cells and for both DNA damage and micronuclei in VERO cells, in a concentration-response relationship. The results of this study indicated that violacein is genotoxic in VERO and FRhK-4 cells. These findings contribute to a comprehensive evaluation of the pharmacological potential of violacein.     

Effect of temperature, pH, and metals on the stability and activity of phenylalanine hydroxylase from Chromobacterium violaceum

Phenylalanine hydroxylase (PAH) is a non-heme iron dioxygenase catalyzing the conversion of phenylalanine to tyrosine and is present in both prokaryotic and eukaryotic organisms. A relatively simple PAH is expressed by Chromobacterium violaceum, a gram-negative bacterium found in tropical and subtropical regions. The effects of temperature, pH and metals on the stability and catalytic activity of Chromobacterium violaceum PAH were determined by steady-state kinetics, circular dichroism (CD) and differential scanning calorimetry (DSC). The kcat and KM for phenylalanine were determined between 7 and 40 degrees C. The KM remained constant between 20 and 40 degrees C but rapidly increased below 20 degrees C. The half-life of the enzyme at 47 degrees C is 66+/-4 min in the presence of Fe(II) and 8+/-1 min in the presence of EDTA. The melting temperature of the protein determined by CD and DSC is 53+/-2 degrees C in the presence of EDTA and 63+/-2 degrees C in the presence of Fe(II). Co(II) stabilizes the enzyme (Tm=63+/-2 degrees C) and inhibits the catalytic activity by displacing iron from the active site. The optimum pH for catalytic activity and stability is 7.4. In conclusion, PAH is adapted for optimal phenylalanine binding at temperatures above 20 degrees C and Fe(II) enhances the resistance of the enzyme to thermal denaturation.     

Formation of indole-3-carboxylic acid by Chromobacterium violaceum.

l-Tryptophan is converted to indole-3-carboxylic acid by growing cultures and resting cell suspensions of Chromobacterium violaceum     

Identification of Chromobacterium violaceum: pigmented and non-pigmented strains.

The classification and, therefore, identification of Chromobacterium violaceum has been based upon its ability to produce a violet pigment. Although the organism may yield non-pigmented variants when subcultured on artificial media, the isolation of non-pigmented strains from pathological tissues or from nature had not been reported. With a method established for the identification of C. violaceum regardless of violet pigmentation, non-pigmented strains were isolated from nature. The presence of non-pigmented strains of C. violaceum in nature is of significance to taxonomy and clinical bacteriology. Pigmentation cannot be held as an essential characteristic of the definition of the genus Chromobacterium and gives credence to the suspicion of Sneath (1960, 1966) that the genus is not a natural one. Non-pigmented strains may have been isolated from clinical material but wrongly identified as belonging to other genera of non-pigmented Gram-negative bacilli and regarded as not being pathogenic.     

Tolerance to stress and environmental adaptability of Chromobacterium violaceum

Chromobacterium violaceum is a Gram-negative bacterium, abundant in a variety of ecosystems in tropical and subtropical regions, including the water and borders of the Negro River, a major component of the Amazon Basin. As a free-living microorganism, C. violaceum is exposed to a series of variable conditions, such as different sources and abundance of nutrients, changes in temperature and pH, toxic compounds and UV rays. These variations, and the wide range of environments, require great adaptability and strong protective systems. The complete genome sequencing of this bacterium has revealed an enormous number and variety of ORFs associated with alternative pathways for energy generation, transport-related proteins, signal transduction, cell motility, secretion, and secondary metabolism. Additionally, the limited availability of iron in most environments can be overcome by iron-chelating compounds, iron-storage proteins, and by several proteins related to iron metabolism in the C. violaceum genome. Osmotically inducible proteins, transmembrane water-channel, and other membrane porins may be regulating the movement of water and maintaining the cell turgor, activities which play an important role in the adaptation to variations in osmotic pressure. Several proteins related to tolerance against antimicrobial compounds, heavy metals, temperature, acid and UV light stresses, others that promote survival under starvation conditions, and enzymes capable of detoxifying reactive oxygen species were also detected in C. violaceum. All these features together help explain its remarkable competitiveness and ability to survive under different types of environmental stress.     

Genotyping of Chromobacterium violaceum isolates by recA PCR-RFLP analysis

Intraspecies variation of Chromobacterium violaceum was examined by comparative sequence - and by restriction fragment length polymorphism analysis of the recombinase A gene (recA-PCR-RFLP). Primers deduced from the known recA gene sequence of the type strain C. violaceum ATCC 12472(T) allowed the specific amplification of a 1040bp recA fragment from each of the 13 C. violaceum strains investigated, whereas other closely related organisms tested negative. HindII-PstI-recA RFLP analysis generated from 13 representative C. violaceum strains enabled us to identify at least three different genospecies. In conclusion, analysis of the recA gene provides a rapid and robust nucleotide sequence-based approach to specifically identify and classify C. violaceum on genospecies level.     

Energetic metabolism of Chromobacterium violaceum

Chromobacterium violaceum is a free-living microorganism, normally exposed to diverse environmental conditions; it has a versatile energy-generating metabolism. This bacterium is capable of exploiting a wide range of energy resources by using appropriate oxidases and reductases. This allows C. violaceum to live in both aerobic and anaerobic conditions. In aerobic conditions, C. violaceum is able to grow in a minimal medium with simple sugars, such as glucose, fructose, galactose, and ribose; both Embden-Meyerhoff, tricarboxylic acid and glyoxylate cycles are used. The respiratory chain supplies energy, as well as substrates for other metabolic pathways. Under anaerobic conditions, C. violaceum metabolizes glucose, producing acetic and formic acid, but not lactic acid or ethanol. C. violaceum is also able to use amino acids and lipids as an energy supply.     

Quorum Sensing Inhibition in Chromobacterium violaceum by Amikacin Combination with Activated Charcoal or Small Plant-Derived Molecules (Pyrogallol and Coumarin)

Microbiology - The study was aimed at effective inhibition of quorum sensing (QS) in the model bacterium Chromobacterium violaceum ATCC 31532 by combinations of compounds with different anti-QS...     

Draft genome sequence of the biocontrol bacterium Chromobacterium sp. strain C-61

Chromobacterium sp. strain C-61 is a plant-associated bacterium with proven capacities to suppress plant diseases. Here, we report the draft genome sequence and automatic annotation of strain C-61. A comparison of this sequence to the sequenced genome of Chromobacterium violaceum ATCC 12472 indicates the novelty of C-61 and a subset of gene functions that may be related to its biocontrol activities.