Isolation and characterization of Escherichia coli phase variants and mutants deficient in type 1 pilus production.

Type 1 pili of Escherichia coli are the prototype of the somatic class of pili found on many strains of bacteria. As a first step in the genetic analysis of type 1 piliation, an extensive series of nonpiliated derivatives of E. coli K-12 strain AW405, was characterized to produce attached or free pili when examined in the antiserum or appeared to produce attached or free pili when examined in the electron microscope. The derivatives fell into two classes; phase variants and mutants. Phase variants that formed colonies of two distinctive types, one associated with a predominantly piliated (P+), and the other associated with a nonpiliated (P-) phase, were obtained. Each phase could give rise to the other at a relatively high rate, which was greater in the P- to P+ direction during culture in unshaken liquid medium. In addition, 77 Pil- mutants were selected on the basis of a subtle difference in colonial morphology. The mutants reverted, if at all, at a much lower rate than that of the P- to P+ change. The stability of Pil- derivatives grown in unshaken liquid medium was used as a criterion for distinguishing between phase variants and mutants, Phase variation also effected colonial morphology and chemotactic swarming. These properties did not directly depend upon piliation since Pil- mutants were only slightly altered in colonial form and unaltered in chemotactic swarming. Piliation of Pil+ bacteria was quantitatively affected by growth conditions.     

Gonococci exit apically and basally from polarized epithelial cells and exhibit dynamic changes in type IV pili

Type IV pili are a major virulence factor of the obligate human pathogen Neisseria gonorrhoeae (the gonococcus; Gc). Pili facilitate bacterial adherence to epithelial cells, but their participation in later steps of epithelial infection, particularly intracellular replication and exit, is poorly understood. Using polarized T84 cells as a model for mature mucosal epithelia, pilus dynamics in piliated, Opa-expressing Gc were examined over time. T84 infection was characterized by a several-hour delay in the growth of cell-associated bacteria and by non-directional exit of Gc, the first time these phenomena have been reported. During infection, non-piliated progeny arose stochastically from piliated progenitors. Piliated and non-piliated Gc replicated and exited from T84 cell monolayers equally well, demonstrating that piliation did not influence Gc survival during epithelial infection. The frequency with which pilin variants arose from a defined piliated progenitor during T84 cell infection was found to be sufficiently high to account for the extensive pilin variation reported during human infection. However, the repertoire of variants appearing in association with T84 cells was similar to what was seen in the absence of cells, demonstrating that polarized epithelial cells can support Gc replication without selecting for a subset of pilin variants or piliation states.     

Scanning electron microscopic study of uropathogen adherence to a plastic surface.

We used a polyethylene surface to study the adherence of various urinary pathogens to a representative inert surface. The bacteria were suspended in filter-sterilized urine during this adhesion study, and differential adhesion was clearly demonstrated. Pseudomonas aeruginosa adhered most avidly and formed large microcolonies that were surrounded by an extensive amorphous matrix. Staphylococcus saprophyticus also formed microcolonies on the surface of the plastic droppers. In general, piliated strains of Escherichia coli adhered less avidly than the other organisms, but more avidly than nonpiliated strains; however, one piliated strain of E. coli adhered very poorly and behaved like a nonpiliated strain.     

Scanning electron microscopic study of uropathogen adherence to a plastic surface

We used a polyethylene surface to study the adherence of various urinary pathogens to a representative inert surface. The bacteria were suspended in filter-sterilized urine during this adhesion study, and differential adhesion was clearly demonstrated. Pseudomonas aeruginosa adhered most avidly and formed large microcolonies that were surrounded by an extensive amorphous matrix. Staphylococcus saprophyticus also formed microcolonies on the surface of the plastic droppers. In general, piliated strains of Escherichia coli adhered less avidly than the other organisms, but more avidly than nonpiliated strains; however, one piliated strain of E. coli adhered very poorly and behaved like a nonpiliated strain.     

Biochemistry of nitrate respiration in Pseudomonas stutzeri. I. Aerobic and nitrate respiration routes of carbohydrate catabolism

Spangler, W. J. (Oregon State University, Corvallis), and C. M. Gilmour. Biochemistry of nitrate respiration in Pseudomonas stutzeri. I. Aerobic and nitrate respiration routes of carbohydrate catabolism. J. Bacteriol. 91:245-250. 1966.-The metabolic pathways of glucose catabolism were studied in Pseudomonas stutzeri under aerobic conditions and under conditions of nitrate respiration. Studies on both glucose and gluconate catabolism, by the radiorespirometric method, indicated that these substrates are degraded in the same manner, i.e., the Entner-Doudoroff and pentose phosphate pathways. There appeared to be no major shift in primary metabolic pathways when nitrate was used as the terminal hydrogen acceptor in nitrate respiration as opposed to aerobic respiration with free molecular oxygen. It was shown that glucose is not degraded to any appreciable extent under anaerobic conditions in the absence of nitrate. Tentative evidence suggests that the tricarboxylic acid cycle functions under both conditions of oxygen relationships and that the rate of carbon oxidation via the tricarboxylic acid cycle is slower with nitrate respiration than under aerobic conditions.     

Factors affecting genetic transformation of Neisseria gonorrhoeae.

Piliated gonococci were competent in genetic transformation in all stages of growth in minimal and enriched media, but nonpiliated cells were almost totally incompetent. Uptake of deoxyribonucleic acid into a deoxyribonuclease-insensitive state was observed only in competent piliated cells. Competence was not affected by washing of competent cells or treatment of competent cells with proteolytic enzymes. Expression of competence required presence of any of several different monovalent or divalent cations, as well as a utilizable source of energy. Efforts to produce genotypically or phenotypically competent derivatives of nonpiliated cells were unsuccessful. These experiments are consistent with the idea that pili may play a role in the irreversible uptake of transforming deoxyribonucleic acid by the gonococcus, but fail to provide evidence for other types of competence factors.     

Construction and Characterization of a Derivative of Neisseria gonorrhoeae Strain MS11 Devoid of All opa Genes

To better understand the role of Opa in gonococcal infections, we created and characterized a derivative of MS11 (MS11Δopa) that had the coding sequence for all 11 Opa proteins deleted. The MS11Δopa bacterium lost the ability to bind to purified lipooligosaccharide (LOS). While nonpiliated MS11Δopa and nonpiliated Opa-expressing MS11 cells grew at the same rate, nonpiliated MS11Δopa cells rarely formed clumps of more than four bacteria when grown in broth with vigorous shaking. Using flow cytometry analysis, we demonstrated that MS11Δopa produced a homogeneous population of bacteria that failed to bind monoclonal antibody (MAb) 4B12, a MAb specific for Opa. Opa-expressing MS11 cells consisted of two predominant populations, where ∼85% bound MAb 4B12 to a significant level and the other population bound little if any MAb. Approximately 90% of bacteria isolated from a phenotypically Opa-negative colony (a colony that does not refract light) failed to bind MAb 4B12; the remaining 10% bound MAb to various degrees. Piliated MS11Δopa cells formed dispersed microcolonies on ME180 cells which were visually distinct from those of piliated Opa-expressing MS11 cells. When Opa expression was reintroduced into MS11Δopa, the adherence ability of the strain recovered to wild-type levels. These data indicate that Opa contributes to both bacterium-bacterium and bacterium-host cell interactions.     

Global Metabolic Response of Enterococcus faecalis to Oxygen

Oxygen and oxidative stress have become relevant components in clarifying the mechanism that weakens bacterial cells in parallel to the mode of action of bactericidal antibiotics. Given the importance of oxidative stress in the overall defense mechanism of bacteria and their apparent role in the antimicrobial mode of action, it is important to understand how bacteria respond to this stress at a metabolic level. The aim of this study was to determine the impact of oxygen on the metabolism of the facultative anaerobe Enterococcus faecalis using continuous culture, metabolomics, and ¹³C enrichment of metabolic intermediates. When E. faecalis was rapidly transitioned from anaerobic to aerobic growth, cellular metabolism was directed toward intracellular glutathione production and glycolysis was upregulated 2-fold, which increased the supply of critical metabolite precursors (e.g., glycine and glutamate) for sulfur metabolism and glutathione biosynthesis as well as reducing power for cellular respiration in the presence of hemin. The ultimate metabolic response of E. faecalis to an aerobic environment was the upregulation of fatty acid metabolism and benzoate degradation, which was linked to important changes in the bacterial membrane composition as evidenced by changes in membrane fatty acid composition and the reduction of membrane-associated demethylmenaquinone. These key metabolic pathways associated with the response of E. faecalis to oxygen may represent potential new targets to increase the susceptibility of this bacterium to bactericidal drugs.     

Respiration rate, growth rate and the accumulation of streptomycin in Escherichia coli

Using chemostat cultures of Escherichia coli it was possible to vary respiration rates while maintaining a constant growth rate. This allowed the effect of variations in respiration rates on the accumulation of streptomycin to be studied in cultures at constant growth rates. At a particular dilution rate cultures exhibited higher respiration rates when phosphate limited growth than when carbon limited growth. A ubiquinone-deficient strain had a lower rate of respiration at a particular dilution rate than a related ubiquinone-sufficient strain. In spite of these differences in respiratory activity, the accumulation of streptomycin was identical in carbon- and in phosphate-limited chemostat cultures of ubiquinone-deficient and ubiquinone-sufficient strains. Moreover, accumulation of streptomycin in an anaerobic chemostat culture occurred at the same rate as that in an aerobic chemostat. There was however a lag of 1.5 h before accumulation commenced in the anaerobic culture, a feature that was not apparent in the aerobic culture. These results indicate that the lower rates of respiration in slow-growing bacteria are not responsible for the decreased accumulation of streptomycin in slow-growing compared to fast-growing cultures. Moreover, it seems unlikely that quinones are involved directly (e.g. as carriers) in streptomycin accumulation, since removal of 90% of cellular ubiquinone, or replacement of ubiquinone with a structural analogue, did not affect accumulation as long as mutant and parent cultures grew at the same rate.     

The Neisseria gonorrhoeae type IV pilus promotes resistance to hydrogen peroxide- and LL-37-mediated killing by modulating the availability of intracellular,labile iron

The Neisseria gonorrhoeae Type IV pilus is a multifunctional, dynamic fiber involved in host cell attachment, DNA transformation, and twitching motility. We previously reported that the N. gonorrhoeae pilus is also required for resistance against hydrogen peroxide-, antimicrobial peptide LL-37-, and non-oxidative, neutrophil-mediated killing. We tested whether the hydrogen peroxide, LL-37, and neutrophil hypersensitivity phenotypes in non-piliated N. gonorrhoeae could be due to elevated iron levels. Iron chelation in the growth medium rescued a nonpiliated pilE mutant from both hydrogen peroxide- and antimicrobial peptide LL-37-mediated killing, suggesting these phenotypes are related to iron availability. We used the antibiotic streptonigrin, which depends on free cytoplasmic iron and oxidation to kill bacteria, to determine whether piliation affected intracellular iron levels. Several non-piliated, loss-of-function mutants were more sensitive to streptonigrin killing than the piliated parental strain. Consistent with the idea that higher available iron levels in the under- and non-piliated strains were responsible for the higher streptonigrin sensitivity, iron limitation by desferal chelation restored resistance to streptonigrin in these strains and the addition of iron restored the sensitivity to streptonigrin killing. The antioxidants tiron and dimethylthiourea rescued the pilE mutant from streptonigrin-mediated killing, suggesting that the elevated labile iron pool in non-piliated bacteria leads to streptonigrin-dependent reactive oxygen species production. These antioxidants did not affect LL-37-mediated killing. We confirmed that the pilE mutant is not more sensitive to other antibiotics showing that the streptonigrin phenotypes are not due to general bacterial envelope disruption. The total iron content of the cell was unaltered by piliation when measured using ICP-MS suggesting that only the labile iron pool is affected by piliation. These results support the hypothesis that piliation state affects N. gonorrhoeae iron homeostasis and influences sensitivity to various host-derived antimicrobial agents.     

Microbial degradation of chlorinated benzenes

Chlorinated benzenes are important industrial intermediates and solvents. Their widespread use has resulted in broad distribution of these compounds in the environment. Chlorobenzenes (CBs) are subject to both aerobic and anaerobic metabolism. Under aerobic conditions, CBs with four or less chlorine groups are susceptible to oxidation by aerobic bacteria, including bacteria (Burkholderia, Pseudomonas, etc.) that grow on such compounds as the sole source of carbon and energy. Sound evidence for the mineralization of CBs has been provided based on stoichiometric release of chloride or mineralization of (14)C-labeled CBs to (14)CO(2). The degradative attack of CBs by these strains is initiated with dioxygenases eventually yielding chlorocatechols as intermediates in a pathway leading to CO(2) and chloride. Higher CBs are readily reductively dehalogenated to lower chlorinated benzenes in anaerobic environments. Halorespiring bacteria from the genus Dehalococcoides are implicated in this conversion. Lower chlorinated benzenes are less readily converted, and mono-chlorinated benzene is recalcitrant to biotransformation under anaerobic conditions.     

胆道手术患者胆汁的菌群分析

本文对59例胆道手术患者的胆汁标本进行了菌群分析。共检出各类细菌156株,其中厌氧菌70株,以类杆菌最为常见(44株);兼性需氧菌86株,以大肠杆菌最为常见(37株)。胆汁细菌培养阳性率为86.4％(51/59);64.4％(38/59)的标本为厌氧菌与兼性厌氧菌混合污染。22.0％(13/59)的标本中仅分离到兼性需氧菌。提示,胆汁中污染的类杆菌及大肠杆菌是胆道手术后感染的主要原因菌。     

Identification and characterization of a gene product that regulates type 1 piliation in Escherichia coli.

The recombinant plasmid pSH2 confers type 1 piliation (Pil+) on a nonpiliated (Pil-) strain of Escherichia coli K-12. At least four plasmid-encoded gene products are involved in pilus biosynthesis and expression. We present evidence which indicates that one gene encodes an inhibitor of piliation. Hyperpiliated (Hyp) mutants were isolated after Tn5 insertion mutagenesis of pSH2 and introduction of the plasmid DNA into a Pil- strain of E. coli as unique small, compact colonies. Also, Hyp mutants clumped during growth in static broth and were piliated under several cultural conditions that normally suppressed piliation. Electron microscopic examination of Hyp mutants associated an observed 40-fold increase in pilin antigen with an increase in the number and length of pili per cell. All Hyp mutants examined failed to produce a 23-kilodalton protein that was encoded by a gene adjacent to the structural (pilin) gene for type 1 pili, and all Tn5 insertion mutations that produced the Hyp phenotype mapped in this region (hyp). Piliation in Hyp mutants could be reduced to near parental levels by introducing a second plasmid containing a parental hyp gene. Thus the 23-kilodalton (hyp) protein appears to act in trans to regulate the level of piliation.     

鲫幼鱼游泳运动能力的个体变异与表型关联

为考察鲤科鱼类运动能力的个体变异和表型关联及不同加速度对匀加速游泳能力的影响, 研究在(25±0.5)℃条件下测定鲫(Carassius auratus)幼鱼的静止代谢率(Resting metabolic rate, RMR), 通过临界游泳速度(Critical swimming speed, U_crit)法和过量耗氧(EPOC)法获取实验鱼的最大代谢率(Maximum metabolic rate, MMR)、代谢空间(Aerobic scope, AS=MMR-RMR)、相对代谢空间(Factorial aerobic scope, FAS=MMR/RMR)、U_crit及步法转换速度(Gait transition speed, U_gt), 并在不同加速度(0.083、0.167、0.250、0.333 cm/s2)下测定鲫幼鱼的匀加速游泳能力(Constant accelerated test, U_cat)和U_gt。研究发现: 鲫幼鱼的MMR和AS与U_crit均呈正相关, 但RMR与U_crit不相关; 能量代谢参数(MMR、AS、RMR)与U_gt不相关。U_crit法获取的MMR、AS、FAS与EPOC法均无平均值的显著性差异, 但2种方法获得的上述参数具有较高的个体重复性; 鲫幼鱼的能量代谢参数之间存在表型关联并且关联方向不尽相同。鲫幼鱼的U_crit和U_gt均小于各加速度下的U_cat和U_gt, 加速度对U_cat测定无影响但对U_gt有影响。鲫幼鱼的U_gt与U_crit或U_cat呈正相关, 并且其匀加速游泳能力参数在不同加速度下保持较高的重复性。除0.333 cm/s2外, 其他加速度下鲫幼鱼U_cat的无氧代谢组分(U_cat-U_gt)与U_cat呈正相关; 然而, 鲫幼鱼的有氧代谢组分(U_gt)与无氧代谢组分(U_cat-U_gt)呈负相关。研究表明: U_crit法和EPOC法诱导鲫幼鱼的有氧代谢能力无方法学差异; 鲫幼鱼的能量代谢存在表型关联, 其匀加速游泳能力具有稳定个体差异, 并且该种鱼的有氧代谢与无氧代谢存在权衡。     

Microbial degradation of chlorinated phenols

Chlorophenols have been introduced into the environment through their use as biocides and as by-products of chlorine bleaching in the pulp and paper industry. Chlorophenols are subject to both anaerobic and aerobic metabolism. Under anaerobic conditions, chlorinated phenols can undergo reductive dechlorination when suitable electron-donating substrates are available. Halorespiring bacteria are known which can use both low and highly chlorinated congeners of chlorophenol as electron acceptors to support growth. Many strains of halorespiring bacteria have the capacity to eliminate ortho-chlorines; however only bacteria from the species Desulfitobacterium hafniense (formerly frappieri) can eliminate para- and meta-chlorines in addition to ortho-chlorines. Once dechlorinated, the phenolic carbon skeletons are completely converted to methane and carbon dioxide by other anaerobic microorganisms in the environment. Under aerobic conditions, both lower and higher chlorinated phenols can serve as sole electron and carbon sources supporting growth. The best studied strains utilizing pentachlorophenol belong to the genera Mycobacterium and Sphingomonas. Two main strategies are used by aerobic bacteria for the degradation of chlorophenols. Lower chlorinated phenols for the most part are initially attacked by monooxygenases yielding chlorocatechols as the first intermediates. On the other hand, polychlorinated phenols are converted to chlorohydroquinones as the initial intermediates. Fungi and some bacteria are additionally known that cometabolize chlorinated phenols.     

Ex situ slurry phase bioremediation of chrysene contaminated soil with the function of metabolic function: process evaluation by data enveloping analysis (DEA) and Taguchi design of experimental methodology (DOE)

Bioremediation of chrysene in soil matrix was evaluated in soil slurry phase bioreactor in conjugation with metabolic functions (aerobic, anoxic and anaerobic), microenvironment (single and mixed) conditions and nature of mixed consortia (native/resident mixed microflora and bioaugmented inoculum). Twelve experiments were operated independently in agitated-batch reactor keeping all other operating conditions constant (substrate loading rate--0.084 g chrysene/kg soil-day; soil loading rate--10 kg soil/m(3)-day (3:25 soil water ratio); operating temperature--35+/-2 degrees C). Data envelopment analysis (DEA) procedure was employed to analyze the performance of experimental variations in terms of chrysene degradation and pH. The efficacy of anoxic metabolism over the corresponding aerobic and anaerobic metabolic functions was documented. Aerobic metabolic function showed effective degradation capability under mixed microenvironment after augmentation with anaerobic inoculum. Anaerobic metabolic function showed lowest degradation potential. Application of bioaugmentation showed positive influence on the chrysene degradation rate. Design of experimental methodology (DOE) by Taguchi approach was applied to evaluate the effect of four selected factors (native soil microflora, microenvironment, metabolic function and bioaugmentation) on the chrysene degradation process. The optimized factors derived from analysis depicted the requirement of native soil microflora under anoxic metabolic function using mixed microenvironment after augmenting with anaerobic inoculum for achieving effective chrysene degradation efficacy.     

乳酸乳球菌双组分系统调控有氧呼吸的研究

【背景】乳酸乳球菌作为食品行业的代表性菌株,如何通过双组分系统响应环境因子与代谢调控的分子机制研究,对发酵食品产业和益生菌制剂行业有着重要的意义。【目的】探究乳酸乳球菌双组分系统对有氧呼吸代谢调控的相关网络,为乳酸菌适应性代谢研究提供新思路。【方法】采用生物信息学方法,系统性地分析乳酸乳球菌双组分系统组氨酸激酶和反应调节因子的结构域组成及预测双组分系统功能,筛选出与有氧呼吸有潜在联系的双组分,并进一步通过基因转录表达和非靶向代谢组学验证。【结果】以乳酸乳球菌的代表菌株NZ9000为例构建相互作用蛋白网络,显示双组分系统与丙酮酸代谢网络关键连接点为丙酮酸铁氧还蛋白氧化还原酶(nifJ)。在不同的生长时期,Lactococcus lactis NZ9000双组分转录表达在延滞期变化显著。与厌氧培养相比,有氧培养和有氧呼吸培养的菌体双组分呈现下调趋势。双组分系统参与乳酸菌氧化应激和血红素胁迫过程。【结论】明确乳酸乳球菌参与有氧呼吸的双组分系统以及代谢通路,有助于提高发酵剂、益生菌剂的存活率和竞争力。     

Transformation of 2,4,6-trinitrotoluene during oxygen and nitrate respiration in Pseudomonas fluorescens

A study of the metabolic pathway and the rate of 2,4,6-trinitrotoluene (TNT) transformation depending on the nature of the electron acceptor in the electron transport chain of Pseudomonas fluorescens B-3468 revealed that the first reaction of nitroreduction of TNT resulting in formation of 2-amino-4,6-dinitrotoluene (2A) and 4-amino-2,6-dinitrotoluene (4A) became more active in case of nitrate respiration as compared to oxygen respiration; a TNT decrease was 100 and 66%, respectively. The same tendency but much more pronounced was observed at the next stage of nitroreduction that lead to 2,4-diamino-6-nitrotoluene (2,4DA). On the contrary, aerobic conditions are more preferable for the subsequent destruction of 2,4DA. Thus monoamino derivatives, 2A and 4A, predominated under anaerobic conditions, whereas 2,4DA under anaerobic ones (85 and 69% of the total nitrogen-containing metabolites), respectively. Phloroglucinol and pyrogallol accumulated in the culture liquid when the bacteria were grown on a medium containing 2,4DA as a sole source of nitrogen. Their role as intermediates was proved by the results obtained by studying oxidative activity of the cells grown in the presence of 2,4DA and phloroglucinol.     

Carbohydrate metabolism in the isolated perfused rat kidney

1. Anaerobic formation of lactate from glucose by isolated perfused rat kidney (411mumol/h per g dry wt.) was three times as fast as in aerobic conditions (138mumol/h per g). 2. In aerobic or in anaerobic conditions, the ratio of lactate production to glucose utilization was about 2. 3. Starvation or acidosis caused a decline of about 30% in the rate of aerobic glycolysis. 4. The rate of formation of glucose from lactate by perfused kidney from a well-fed rat, in the presence of 5mm-acetoacetate (83mumol/h per g dry wt.), was of the same order as the rate of aerobic glycolysis. 5. During perfusion with physiological concentrations of glucose (5mm) and lactate (2mm) there were negligible changes in the concentration of either substrate. 6. Comparison of kidneys perfused with lactate, from well-fed or starved rats, showed no major differences in contents of intermediates of gluconeogenesis. 7. The tissue concentrations of hexose monophosphates and C(3) phosphorylated glycolytic intermediates (except triose phosphate) were decreased in anaerobic conditions. 8. Aerobic metabolism of fructose by perfused kidney was rapid: the rate of glucose formation was 726mumol/h per g dry wt. and of lactate formation 168mumol/h per g (dry wt.). Glycerol and d-glyceraldehyde were also released into the medium. 9. Aerobically, fructose generated high concentrations of glycolytic intermediates. 10. Anaerobic production of lactate from fructose (74mumol/h per g dry wt.) was slower than the aerobic rate. 11. In both anaerobic and aerobic conditions the ratio [lactate]/[pyruvate] in kidney or medium was lower during perfusion with fructose than with glucose. 12. These results are discussed in terms of the regulation of renal carbohydrate metabolism.