Nonlinearity in the Growth of Bacterial Colonies: Conditions and Causes

The universally recognized kinetic model of colony growth, introduced by Pirt, predicts a linear increase of colony size. The linearity follows from the assumption that the colony expands through the growth of only such cells that are located immediately behind the moving colony front, in the so-called peripheral zone of constant width and density. In this work, Pirt's model was tested on two bacteria—Alcaligenes sp. and Pseudomonas fluorescens—having markedly distinct cultural properties and grown on an agarized medium with pyruvate. The colony size dynamics was followed for different densities of the inoculum, ranging from a single cell to a microdroplet of bacterial suspension (10⁵–10⁶ cells), and for different depths of the agar layer, determining the amount of available substrate. A linear growth mode was observed only with P. fluorescens and only in the case of growth from a microdroplet. When originating from a single cell, colonies of both organisms displayed nonlinear growth with a distinct peak of K _r (the rate of colony radius increase) occurring after 2–3 days of growth. The growth of P. fluorescens colonies showed virtually no dependence on the depth of the agarized medium, whereas the rate of colony size increase of Alcaligenes sp. turned out to be directly related to the medium layer thickness. The departure from linearity is consistently explained by a new kinetic scheme stipulating a possible contribution to the colony growth not only of peripheral cells but also (much more distinct in Alcaligenes) of cells at the colony center. The colony growth dynamics is determined not only by the concentration of the limiting substrate but also by the amount of autoinhibitor, the synthesis of which is governed by the age of cells. The distinctions of growth from a single cell and microdroplet could also originate as a result of dissociation into the R- and S-forms and competition between the corresponding subpopulations for oxygen and the common substrate.     

Utilization of d-Lactate as an Energy Source Supports the Growth of Gluconobacter oxydans

d-Lactate was identified as one of the few available organic acids that supported the growth of Gluconobacter oxydans 621H in this study. Interestingly, the strain used d-lactate as an energy source but not as a carbon source, unlike other lactate-utilizing bacteria. The enzymatic basis for the growth of G. oxydans 621H on d-lactate was therefore investigated. Although two putative NAD-independent d-lactate dehydrogenases, GOX1253 and GOX2071, were capable of oxidizing d-lactate, GOX1253 was the only enzyme able to support the d-lactate-driven growth of the strain. GOX1253 was characterized as a membrane-bound dehydrogenase with high activity toward d-lactate, while GOX2071 was characterized as a soluble oxidase with broad substrate specificity toward d-2-hydroxy acids. The latter used molecular oxygen as a direct electron acceptor, a feature that has not been reported previously in d-lactate-oxidizing enzymes. This study not only clarifies the mechanism for the growth of G. oxydans on d-lactate, but also provides new insights for applications of the important industrial microbe and the novel d-lactate oxidase.     

Staining of Bacterial Colonies on the Millipore Filter to Improve Contrast

About 5 ml of 1% blue tetrazolium in 70% ethyl alcohol were poured over mature colonies of Pasteurella pestis and Malleomyces pseudomallei on Millipore filters (MF), contained in the filter holder apparatus, and allowed to drain through with the suction applied. The MF was washed with water and then covered with about 10 ml of 0.001% aqueous trypan blue and drained. This technique provided vivid white colonies sharply defined against a blue background.

Another method utilized 0.1% quinacrine-HCl (Atabrine) to stain colonies yellow and 0.05% vital red to stain the MF pink to light red.     

Repulsion and Metabolic Switches in the Collective Behavior of Bacterial Colonies

Bacteria inoculated on surfaces create colonies that spread out, forming patterns shaped by their mutual interactions. Here, by a combination of experiments and modeling, we address two striking phenomena observed when colonies spread out circularly, without dendritic instabilities. First, the velocity of spreading is generically found to decrease as levels of nutrients initially deposited on the surface increase. We demonstrate that the slowdown is due to phenomena of differentiation, leading to the coexistence of bacteria in different states of motility and we model their dynamics. Second, colonies spreading out from different inocula on the same surface are observed to merge or repel (halting at a finite distance), depending on experimental conditions. We identify the parameters that determine the fate of merging versus repulsion, and predict the profile of arrest in the cases of repulsion.     

Electron-Microscopic Studies of the Colonies of an Alkylsulfonate-utilizing Bacterial Consortium

The light- and electron-microscopic analysis of the colonies of a bacterial consortium capable of utilizing alkylsulfonates, which are the main ingredients of waste from the synthetic rubber industry, revealed the presence of eight types of cells. All types of cells were gram-negative and differed in shape, size, and the presence of capsule and cytoplasmic inclusions. Three types of cells were present in all the colonies studied. The presence of the other types of cells depended on the inoculum used and on the composition of the growth medium.     

Normal Aging Modulates the Neurotoxicity of Mutant Huntingtin

Aging likely plays a role in neurodegenerative disorders. In Huntington''s disease (HD), a disorder caused by an abnormal expansion of a polyglutamine tract in the protein huntingtin (Htt), the role of aging is unclear. For a given tract length, the probability of disease onset increases with age. There are mainly two hypotheses that could explain adult onset in HD: Either mutant Htt progressively produces cumulative defects over time or “normal” aging renders neurons more vulnerable to mutant Htt toxicity. In the present study, we directly explored whether aging affected the toxicity of mutant Htt in vivo. We studied the impact of aging on the effects produced by overexpression of an N-terminal fragment of mutant Htt, of wild-type Htt or of a β-Galactosidase (β-Gal) reporter gene in the rat striatum. Stereotaxic injections of lentiviral vectors were performed simultaneously in young (3 week) and old (15 month) rats. Histological evaluation at different time points after infection demonstrated that the expression of mutant Htt led to pathological changes that were more severe in old rats, including an increase in the number of small Htt-containing aggregates in the neuropil, a greater loss of DARPP-32 immunoreactivity and striatal neurons as assessed by unbiased stereological counts.The present results support the hypothesis that “normal” aging is involved in HD pathogenesis, and suggest that age-related cellular defects might constitute potential therapeutic targets for HD.     

Bacterial Activity and Bacterioplankton Diversity in the Eutrophic River Warnow��Direct Measurement of Bacterial Growth Efficiency and Its Effect on Carbon Utilization

The influence of bacterial activity and diversity on bacterial growth efficiency was investigated in a flatland river. Eutrophic River Warnow drains predominantly agricultural land and is heavily loaded with nutrients, dissolved and particulate organic matter (DOM and POM), especially humic substances. Although the water column bacterial community consists of many inactive or damaged cells, bacterioplankton sustained a high bacterial secondary production of 0.2?C14.5???g C L^?1 h^?1 and a high DNA synthesis (thymidine uptake) of 6.1?C15.5???g C L^?1 h^?1. The direct and short-term measurement of bacterial respiration (by optodes) revealed high respiration rates especially in summer leading to directly estimated bacterial growth efficiencies (BGE) of 2?C28%. These values are compared to calculations based only on bacterial production, which considerably overestimated BGEs. From all these data, River Warnow can be characterized as a strongly remineralizing system. River Warnow was dominated among others by Cytophaga/Flavobacteria and Actinobacteria which are typical for organic rich waters because of their ability to degrade high molecular weight compounds. However, community composition did not significantly affect BGE.     

Characterisation of a GroEL Single-Ring Mutant that Supports Growth of Escherichia coli and Has GroES-Dependent ATPase Activity

Binding and folding of substrate proteins by the molecular chaperone GroEL alternates between its two seven-membered rings in an ATP-regulated manner. The association of ATP and GroES to a polypeptide-bound ring of GroEL encapsulates the folding proteins in the central cavity of that ring (cis ring) and allows it to fold in a protected environment where the risk of aggregation is reduced. ATP hydrolysis in the cis ring changes the potentials within the system such that ATP binding to the opposite (trans) ring triggers the release of all ligands from the cis ring of GroEL through a complex network of allosteric communication between the rings. Inter-ring allosteric communication thus appears indispensable for the function of GroEL, and an engineered single-ring version (SR1) cannot substitute for GroEL in vivo. We describe here the isolation and characterisation of an active single-ring form of the GroEL protein (SR-A92T), which has an exceptionally low ATPase activity that is strongly stimulated by the addition of GroES. Dissection of the kinetic pathway of the ATP-induced structural changes in this active single ring can be explained by the fact that the mutation effectively blocks progression through the full allosteric pathway of the GroEL reaction cycle, thus trapping an early allosteric intermediate. Addition of GroES is able to overcome this block by binding this intermediate and pulling the allosteric pathway to completion via mass action, explaining how bacterial cells expressing this protein as their only chaperonin are viable.     

Selective Control of Attention Supports the Positivity Effect in Aging

There is emerging evidence for a positivity effect in healthy aging, which describes an age-specific increased focus on positive compared to negative information. Life-span researchers have attributed this effect to the selective allocation of cognitive resources in the service of prioritized emotional goals. We explored the basic principles of this assumption by assessing selective attention and memory for visual stimuli, differing in emotional content and self-relevance, in young and old participants. To specifically address the impact of cognitive control, voluntary attentional selection during the presentation of multiple-item displays was analyzed and linked to participants'' general ability of cognitive control. Results revealed a positivity effect in older adults'' selective attention and memory, which was particularly pronounced for self-relevant stimuli. Focusing on positive and ignoring negative information was most evident in older participants with a generally higher ability to exert top-down control during visual search. Our findings highlight the role of controlled selectivity in the occurrence of a positivity effect in aging. Since the effect has been related to well-being in later life, we suggest that the ability to selectively allocate top-down control might represent a resilience factor for emotional health in aging.     

糖蜜酒精废水的生物脱色及其细菌群落结构分析

糖蜜酒精废水是糖厂利用糖蜜生产酒精后的废液,废水中色素的含量较高,脱色为该类废水处理过程中的一大难题。采用富集培养技术,将取自糖厂IC(internal circulation)反应器底部的活性污泥加入到不同浓度废水中进行富集,得到的菌群可使废水脱色24%、COD(chemical oxygen demand)去除率19.2%。为了提高菌群的脱色能力,研究了几种单因素对菌群生长、脱色及COD去除能力的影响,结果发现,菌群使废水脱色和去除COD的最佳条件为:10%(v/v)废水中添加0.5%葡萄糖、0.1%蛋白胨、0.1%KH2PO4、0.05%MgSO4·7H2O、0.05%KCl,废水初始pH7.0,在37℃培养7d,废水脱色率达46.2%、COD去除率为38.5%。为了了解以上条件下菌群的群落结构,采用分子生态学方法构建了菌群中细菌16S rDNA文库并对代表性的克隆进行了测序,结果显示其中细菌主要分布在Firmicutes、Proteobacteria、Bacteroidetes的7个目中,分别是Erysipelotrich-ales、Clostridiales、Lactobacillales、Xanthomonadales、Burkholderiales、Enterobacteriales、Bacteroidales。大多数细菌属于发酵型细菌,化能异氧型。本研究结果为利用微生物调控糖蜜酒精废水的生物处理提供了依据。     

The Maintenance of Discrete Bacterial Colonies on the Surface of Objects Embedded in Agar

Summary: Bacterial colonies grown on objects embedded in agar sometimes show a tendency to become confluent. Glass coverslips and polytetrafluorethylene (Teflon) discs were inoculated with Staphylococcus lactis and completely embedded in serum agar. If the surface of the agar were flooded with broth before the plates were incubated the colonies had a tendency to run together, but not if the plates were incubated after thorough drying. Drying also prevented confluence occurring when broth had been injected on to the surface of the embedded object. Drying the poured plate probably acts by removing fluid squeezed out at the interface.     

Interplay between Intrinsic Noise and the Stochasticity of the Cell Cycle in Bacterial Colonies

Herein we report on the effects that different stochastic contributions induce in bacterial colonies in terms of protein concentration and production. In particular, we consider for what we believe to be the first time cell-to-cell diversity due to the unavoidable randomness of the cell-cycle duration and its interplay with other noise sources. To that end, we model a recent experimental setup that implements a protein dilution protocol by means of division events to characterize the gene regulatory function at the single cell level. This approach allows us to investigate the effect of different stochastic terms upon the total randomness experimentally reported for the gene regulatory function. In addition, we show that the interplay between intrinsic fluctuations and the stochasticity of the cell-cycle duration leads to different constructive roles. On the one hand, we show that there is an optimal value of protein concentration (alternatively an optimal value of the cell cycle phase) such that the noise in protein concentration attains a minimum. On the other hand, we reveal that there is an optimal value of the stochasticity of the cell cycle duration such that the coherence of the protein production with respect to the colony average production is maximized. The latter can be considered as a novel example of the recently reported phenomenon of diversity induced resonance.     

Novel Perspectives on the Characterization of Species-Dependent Optical Signatures of Bacterial Colonies by Digital Holography

The use of light diffraction for the microbiological diagnosis of bacterial colonies was a significant breakthrough with widespread implications for the food industry and clinical practice. We previously confirmed that optical sensors for bacterial colony light diffraction can be used for bacterial identification. This paper is focused on the novel perspectives of this method based on digital in-line holography (DIH), which is able to reconstruct the amplitude and phase properties of examined objects, as well as the amplitude and phase patterns of the optical field scattered/diffracted by the bacterial colony in any chosen observation plane behind the object from single digital hologram. Analysis of the amplitude and phase patterns inside a colony revealed its unique optical properties, which are associated with the internal structure and geometry of the bacterial colony. Moreover, on a computational level, it is possible to select the desired scattered/diffracted pattern within the entire observation volume that exhibits the largest amount of unique, differentiating bacterial features. These properties distinguish this method from the already proposed sensing techniques based on light diffraction/scattering of bacterial colonies. The reconstructed diffraction patterns have a similar spatial distribution as the recorded Fresnel patterns, previously applied for bacterial identification with over 98% accuracy, but they are characterized by both intensity and phase distributions. Our results using digital holography provide new optical discriminators of bacterial species revealed in one single step in form of new optical signatures of bacterial colonies: digital holograms, reconstructed amplitude and phase patterns, as well as diffraction patterns from all observation space, which exhibit species-dependent features. To the best of our knowledge, this is the first report on bacterial colony analysis via digital holography and our study represents an innovative approach to the subject.     

Prevalence and Correlates of Bacterial Vaginosis in Different Sub-Populations of Women in Sub-Saharan Africa: A Cross-Sectional Study

Background

Clinical development of vaginally applied products aimed at reducing the transmission of HIV and other sexually transmitted infections, has highlighted the need for a better characterisation of the vaginal environment. We set out to characterise the vaginal environment in women in different settings in sub-Saharan Africa.

Methods

A longitudinal study was conducted in Kenya, Rwanda and South-Africa. Women were recruited into pre-defined study groups including adult, non-pregnant, HIV-negative women; pregnant women; adolescent girls; HIV-negative women engaging in vaginal practices; female sex workers; and HIV-positive women. Consenting women were interviewed and underwent a pelvic exam. Samples of vaginal fluid and a blood sample were taken and tested for bacterial vaginosis (BV), HIV and other reproductive tract infections (RTIs). This paper presents the cross-sectional analyses of BV Nugent scores and RTI prevalence and correlates at the screening and the enrolment visit.

Results

At the screening visit 38% of women had BV defined as a Nugent score of 7–10, and 64% had more than one RTI (N. gonorrhoea, C. trachomatis, T. vaginalis, syphilis) and/or Candida. At screening the likelihood of BV was lower in women using progestin-only contraception and higher in women with more than one RTI. At enrolment, BV scores were significantly associated with the presence of prostate specific antigen (PSA) in the vaginal fluid and with being a self-acknowledged sex worker. Further, sex workers were more likely to have incident BV by Nugent score at enrolment.

Conclusions

Our study confirmed some of the correlates of BV that have been previously reported but the most salient finding was the association between BV and the presence of PSA in the vaginal fluid which is suggestive of recent unprotected sexual intercourse.     

Tests of Bacterial Strain Dominance within a Mouse Based on Sampling Faecal Colonies

This paper deals with making inferences about the dominance of a bacterial strain in a mouse gut based on strain typing of a random sample of colonies derived from plated faecal material.     

Inference about the Number of Resident Bacterial Strains when Sampling from Plate Colonies

This paper deals with making inferences about the number of resident bacterial strains in mice gut based on the number of strains of bacteria found in a random sample of colonies derived from plated faecal material. It is shown that for what seems to be a natural way to test the hypothesis that there is only one resident bacterial strain against the alternative that there is more than one resident strain the probability of a Type II error may be unacceptably large. It is also shown that given the sample contains only one bacterial strain the probability that the animal has only one resident strain converges to unity at a geometric rate as the sample size increases.