Indirect and suboptimal control of gene expression is widespread in bacteria

Gene regulation in bacteria is usually described as an adaptive response to an environmental change so that genes are expressed when they are required. We instead propose that most genes are under indirect control: their expression responds to signal(s) that are not directly related to the genes’ function. Indirect control should perform poorly in artificial conditions, and we show that gene regulation is often maladaptive in the laboratory. In Shewanella oneidensis MR‐1, 24% of genes are detrimental to fitness in some conditions, and detrimental genes tend to be highly expressed instead of being repressed when not needed. In diverse bacteria, there is little correlation between when genes are important for optimal growth or fitness and when those genes are upregulated. Two common types of indirect control are constitutive expression and regulation by growth rate; these occur for genes with diverse functions and often seem to be suboptimal. Because genes that have closely related functions can have dissimilar expression patterns, regulation may be suboptimal in the wild as well as in the laboratory.     

Stress-induced mutagenesis in bacteria

Bacteria spend their lives buffeted by changing environmental conditions. To adapt to and survive these stresses, bacteria have global response systems that result in sweeping changes in gene expression and cellular metabolism. These responses are controlled by master regulators, which include: alternative sigma factors, such as RpoS and RpoH; small molecule effectors, such as ppGpp; gene repressors such as LexA; and, inorganic molecules, such as polyphosphate. The response pathways extensively overlap and are induced to various extents by the same environmental stresses. These stresses include nutritional deprivation, DNA damage, temperature shift, and exposure to antibiotics. All of these global stress responses include functions that can increase genetic variability. In particular, up-regulation and activation of error-prone DNA polymerases, down-regulation of error-correcting enzymes, and movement of mobile genetic elements are common features of several stress responses. The result is that under a variety of stressful conditions, bacteria are induced for genetic change. This transient mutator state may be important for adaptive evolution.     

Stress-Induced Mutagenesis in Bacteria

ABSTRACT

Bacteria spend their lives buffeted by changing environmental conditions. To adapt to and survive these stresses, bacteria have global response systems that result in sweeping changes in gene expression and cellular metabolism. These responses are controlled by master regulators, which include: alternative sigma factors, such as RpoS and RpoH; small molecule effectors, such as ppGpp; gene repressors such as LexA; and, inorganic molecules, such as polyphosphate. The response pathways extensively overlap and are induced to various extents by the same environmental stresses. These stresses include nutritional deprivation, DNA damage, temperature shift, and exposure to antibiotics. All of these global stress responses include functions that can increase genetic variability. In particular, up-regulation and activation of error-prone DNA polymerases, down-regulation of error-correcting enzymes, and movement of mobile genetic elements are common features of several stress responses. The result is that under a variety of stressful conditions, bacteria are induced for genetic change. This transient mutator state may be important for adaptive evolution.     

Modeling the electron transport chain of purple non‐sulfur bacteria

Purple non‐sulfur bacteria (Rhodospirillaceae) have been extensively employed for studying principles of photosynthetic and respiratory electron transport phosphorylation and for investigating the regulation of gene expression in response to redox signals. Here, we use mathematical modeling to evaluate the steady‐state behavior of the electron transport chain (ETC) in these bacteria under different environmental conditions. Elementary‐modes analysis of a stoichiometric ETC model reveals nine operational modes. Most of them represent well‐known functional states, however, two modes constitute reverse electron flow under respiratory conditions, which has been barely considered so far. We further present and analyze a kinetic model of the ETC in which rate laws of electron transfer steps are based on redox potential differences. Our model reproduces well‐known phenomena of respiratory and photosynthetic operation of the ETC and also provides non‐intuitive predictions. As one key result, model simulations demonstrate a stronger reduction of ubiquinone when switching from high‐light to low‐light conditions. This result is parameter insensitive and supports the hypothesis that the redox state of ubiquinone is a suitable signal for controlling photosynthetic gene expression.     

Isolation of Optically Targeted Single Bacteria by Application of Fluidic Force Microscopy to Aerobic Anoxygenic Phototrophs from the Phyllosphere

In their natural environment, bacteria often behave differently than they do under laboratory conditions. To gain insight into the physiology of bacteria in situ, dedicated approaches are required to monitor their adaptations and specific behaviors under environmental conditions. Optical microscopy is crucial for the observation of fundamental characteristics of bacteria, such as cell shape, size, and marker gene expression. Here, fluidic force microscopy (FluidFM) was exploited to isolate optically selected bacteria for subsequent identification and characterization. In this study, bacteriochlorophyll-producing bacteria, which can be visualized due to their characteristic fluorescence in the infrared range, were isolated from leaf washes. Bacterial communities from the phyllosphere were investigated because they harbor genes indicative of aerobic anoxygenic photosynthesis. Our data show that different species of Methylobacterium express their photosystem in planta, and they show a distinct pattern of bacteriochlorophyll production under laboratory conditions that is dependent on supplied carbon sources.     

The NtrY/X two-component system of Brucella spp. acts as a redox sensor and regulates the expression of nitrogen respiration enzymes

Brucella spp. are facultative intracellular bacteria pathogenic for many mammalian species including humans, causing a disease called brucellosis. Learning how Brucella adapts to its intracellular niche is crucial for understanding its pathogenesis mechanism, allowing for the development of new and more effective vaccines and treatments against brucellosis. Brucella pathogenesis resides mostly in its ability to adapt to the harsh environmental conditions encountered during host infection such as the oxygen depletion. The mechanism by which Brucella senses the oxygen tension and triggers its environmental adaptation is unknown. In this work we show that the Brucella abortus NtrY/NtrX two-component system is involved in oxygen sensing through a haem group contained in a Per-ARNT-SIM (PAS) domain of the NtrY histidine kinase. The NtrY haem iron can be reduced to the ferrous form and is rapidly oxidized to the ferric form in presence of oxygen. Importantly, we show that the oxidation state of the haem iron modulates the autokinase activity, being the anoxygenic reduced ferrous form the signalling state of NtrY. Also, we show that ntrY gene expression increases under low oxygen tension and that NtrY transfers its signal to its cognate response regulator NtrX, regulating in this way the expression of nitrogen respiration enzymes. Based on these findings, we postulate that NtrY acts as a redox sensor in Brucella spp.     

Involvement of rpoS in the survival of Escherichia coli in the viable but non-culturable state

When exposed to stress-provoking environmental conditions such as those of ground waters, many medically important bacteria have been shown to be capable of activating a survival strategy known as the viable but non-culturable (VBNC) state. In this state bacteria are no longer culturable on conventional growth media, but the cells maintain their viability and pathogenicity genes/factors and can start dividing again, in a part of the cell population, upon restoration of favourable environmental conditions. Little is known about the genetic mechanisms underlying the VBNC state. In this study we show evidence of involvement of the rpoS gene in persistence of Escherichia coli in the VBNC state. The kinetics of entry into the non-culturable state and duration of cell viability were measured in two E. coli mutants carrying an inactivated rpoS gene and compared with those of the parents. For these experiments, laboratory microcosms consisting of an artificial oligotrophic medium incubated at 4 degrees C were used. The E. coli parental strains reached the non-culturable state in 33 days when the plate counts were evaluated on Luria-Bertani agar containing sodium pyruvate, whereas cells of the rpoS mutants lost their culturability in only 21 days. Upon reaching unculturability the parents yielded respiring cells and cells with intact membranes for at least the next three weeks and resuscitation was allowed during this time. In contrast, the RpoS- mutant cells demonstrated intact membranes for only two weeks and a very restricted (<7 days) resuscitation capability. Guanosine 3',5'-bispyrophosphate (ppGpp) acts as a positive regulator during the production and functioning of RpoS. A mutant deficient in ppGpp production behaved like the rpoS mutants, while overproducers of ppGpp displayed a vitality at least comparable to that of RpoS+ strains. These results suggest that the E. coli parental strains enter the VBNC state which lasts for, at least, three weeks, after which apparently all the cells die. The rpoS mutants do not activate this survival strategy and early die. This implies involvement of the rpoS gene in E. coli persistence in the VBNC state.     

Nongenetic Individuality in the Host–Phage Interaction

Isogenic bacteria can exhibit a range of phenotypes, even in homogeneous environmental conditions. Such nongenetic individuality has been observed in a wide range of biological processes, including differentiation and stress response. A striking example is the heterogeneous response of bacteria to antibiotics, whereby a small fraction of drug-sensitive bacteria can persist under extensive antibiotic treatments. We have previously shown that persistent bacteria enter a phenotypic state, identified by slow growth or dormancy, which protects them from the lethal action of antibiotics. Here, we studied the effect of persistence on the interaction between Escherichia coli and phage lambda. We used long-term time-lapse microscopy to follow the expression of green fluorescent protein (GFP) under the phage lytic promoter, as well as cellular fate, in single infected bacteria. Intriguingly, we found that, whereas persistent bacteria are protected from prophage induction, they are not protected from lytic infection. Quantitative analysis of gene expression reveals that the expression of lytic genes is suppressed in persistent bacteria. However, when persistent bacteria switch to normal growth, the infecting phage resumes the process of gene expression, ultimately causing cell lysis. Using mathematical models for these two host–phage interactions, we found that the bacteria's nongenetic individuality can significantly affect the population dynamics, and might be relevant for understanding the coevolution of bacterial hosts and phages.     

Nongenetic individuality in the host-phage interaction

Isogenic bacteria can exhibit a range of phenotypes, even in homogeneous environmental conditions. Such nongenetic individuality has been observed in a wide range of biological processes, including differentiation and stress response. A striking example is the heterogeneous response of bacteria to antibiotics, whereby a small fraction of drug-sensitive bacteria can persist under extensive antibiotic treatments. We have previously shown that persistent bacteria enter a phenotypic state, identified by slow growth or dormancy, which protects them from the lethal action of antibiotics. Here, we studied the effect of persistence on the interaction between Escherichia coli and phage lambda. We used long-term time-lapse microscopy to follow the expression of green fluorescent protein (GFP) under the phage lytic promoter, as well as cellular fate, in single infected bacteria. Intriguingly, we found that, whereas persistent bacteria are protected from prophage induction, they are not protected from lytic infection. Quantitative analysis of gene expression reveals that the expression of lytic genes is suppressed in persistent bacteria. However, when persistent bacteria switch to normal growth, the infecting phage resumes the process of gene expression, ultimately causing cell lysis. Using mathematical models for these two host–phage interactions, we found that the bacteria's nongenetic individuality can significantly affect the population dynamics, and might be relevant for understanding the coevolution of bacterial hosts and phages.     

Effects of population density and chemical environment on the behavior of Escherichia coli in shallow temperature gradients

In shallow temperature gradients, changes in temperature that bacteria experience occur over long time scales. Therefore, slow processes such as adaptation, metabolism, chemical secretion and even gene expression become important. Since these are cellular processes, the cell density is an important parameter that affects the bacteria's response. We find that there are four density regimes with distinct behaviors. At low cell density, bacteria do not cause changes in their chemical environment; however, their response to the temperature gradient is strongly influenced by it. In the intermediate cell-density regime, the consumption of nutrients becomes significant and induces a gradient of nutrients opposing the temperature gradient due to higher consumption rate at the high temperature. This causes the bacteria to drift toward low temperature. In the high cell-density regime, interactions among bacteria due to secretion of an attractant lead to a strong local accumulation of bacteria. This together with the gradient of nutrients, resulted from the differential consumption rate, creates a fast propagating pulse of bacterial density. These observations are a result of classical nonlinear population dynamics. At extremely high cell density, a change in the physiological state of the bacteria is observed. The bacteria, at the individual level, become cold seeking. This appears initially as a result of a change in the methylation level of the two most abundant sensing receptors, Tsr and Tar. It is further enforced at an even higher cell density by a change in the expression level of these receptors.     

Correlation between bacterial haemoglobin gene (vgb) and aeration: their effect on the growth and alpha-amylase activity in transformed Enterobacter aerogenes

AIMS: To evaluate the effects of bacterial haemoglobin on bacterial growth and alpha-amylase formation under different aeration conditions. METHODS AND RESULTS: Enterobacter aerogenes was transformed with the gene encoding Vitreoscilla (bacterial) haemoglobin, vgb. The growth kinetics and ability to synthesize alpha-amylase enzyme were investigated in this transformed Enterobacter strain as well as in two other Enterobacter control strains that do not harbour the vgb gene. Such comparison was made under variable aeration conditions, using the agitation rate as a measure of aeration. The expression of bacterial haemoglobin-supported cell growth determined as O.D.600 and cell viability in addition to the alpha-amylase production. These positive effects of bacterial haemoglobin were observed under both low and high aerations, but at different extents. CONCLUSIONS: In addition to improving cell growth under low aeration, the bacterial haemoglobin is able to promote bacterial cell tolerance during exposure to high oxygen tension. SIGNIFICANCE AND IMPACT OF THE STUDY: The expression of bacterial haemoglobin is advantageous in reducing the burden of certain toxic conditions such as high oxygen levels. It may have the same impact on some environmental toxic substances. This, haemoglobin biotechnology can be extended to induce enzymes of pollutants degradation or production of some useful industrial substances.     

Application of polyphosphate metabolism to environmental and biotechnological problems

The synthesis and degradation of polyphosphate (polyP) are influenced by the energy state of the cell and extracellular phosphate levels. The import of excess phosphate and its incorporation into polyP under phosphate- and energy-rich growth conditions allows organisms to survive when phosphate or energy are depleted. Under phosphate-starvation conditions, phosphate can be recovered from polyP by hydrolysis. When the organism is energy starved, energy can be recovered either by regenerating the high-energy phosphoanhydride bond donor (ATP in most cases) or by hydrolysis of polyP and subsequent secretion of orthophosphate to recharge the transmembrane proton gradient. Understanding how the energy state of the cell and environmental phosphate levels affect polyP metabolism is essential to improving such environmental processes as enhanced biological phosphorus removal, a treatment process that is widely used to remove excess phosphate from wastewater. Manipulation of the genes responsible for polyP metabolism can also be used to improve gene expression from phosphate-starvation promoters and to remove heavy metals from contaminated environments.     

Fate of Pathogenic Bacteria in Microcosms Mimicking Human Body Sites

During the infectious process, pathogens may reach anatomical sites where they are exposed to substances interfering with their growth. These substances can include molecules produced by the host, and his resident microbial population, as well as exogenous antibacterial drugs. Suboptimal concentrations of inhibitory molecules and stress conditions found in vivo (high or low temperatures, lack of oxygen, extreme pH) might induce in bacteria the activation of survival mechanisms blocking their division capability but allowing them to stay alive. These “dormant” bacteria can be reactivated in particular circumstances and would be able to express their virulence traits. In this study, it was evaluated the effect of some environmental conditions, such as optimal and suboptimal temperatures, direct light and antibiotic sub-inhibitory concentrations doses of antibiotic, on the human pathogens Escherichia coli and Enterococcus faecalis when incubated in fluids accumulated in the body of patients with different pathologies. It is shown that inoculation in a number of accumulated body fluids and the presence of gentamicin, reliable conditions encountered during pathological states, induce stress-responding strategies enabling bacteria to persist in microcosms mimicking the human body. Significant differences were detected in Gram-negative and Gram-positive species with E. faecalis surviving, as starved or viable but non-culturable forms, in any microcosm and condition tested and E. coli activating a viable but non-culturable state only in some clinical samples. The persistence of bacteria under these conditions, being non-culturable, might explain some recurrent infections without isolation of the causative agent after application of the standard microbiological methods.     

Environmental and genetic regulation of the phosphorylcholine epitope of Haemophilus influenzae lipooligosaccharide

In response to environmental signals in the host, bacterial pathogens express factors required during infection and repress those that interfere with specific stages of this process. Signalling pathways controlling virulence factors of the human respiratory pathogen, Haemophilus influenzae, are predominantly unknown. The lipooligosaccharide (LOS) outer core represents a prototypical virulence trait of H. influenzae that enhances virulence but also provides targets for innate and adaptive immunity. We report regulation of the display of the virulence-associated phosphorylcholine (PC) epitope on the LOS in response to environmental conditions. PC display is optimal under microaerobic conditions and markedly decreased under conditions of high culture aeration. Gene expression analysis using a DNA microarray was performed to begin to define the metabolic state of the cell under these conditions and to identify genes potentially involved in PC epitope modulation. Global gene expression profiling detected changes in redox responsive genes and in genes of carbohydrate metabolism. The effects on carbohydrate metabolism led us to examine the role of the putative H. influenzae homologue of csrA, a regulator of glycolysis and gluconeogenesis in Escherichia coli. A mutant containing an in-frame deletion of the H. influenzae csrA gene showed increased PC epitope levels under aerobic conditions. Furthermore, deletion of csrA elevated mRNA expression of galU, an essential virulence gene that is critical in generating sugar precursors needed for polysaccharide formation and LOS outer core synthesis. Growth conditions predicted to alter the redox state of the culture modulated the PC epitope and galU expression as well. The results are consistent with a multifactorial mechanism of control of LOS-PC epitope display involving csrA and environmental signals that coordinately regulate biosynthetic and metabolic genes controlling the LOS structure.