The human urobiome.

Traditionally, the healthy urinary bladder has been considered to be sterile. Several teams have used metagenomic (DNA-dependent) and metaculturomic (culture-dependent) methods to debunk this longstanding dogma. In fact, resident microbial communities (urobiome) have been detected in both adult females and males. Although the field is young, several observations have been made. For example, the urobiome differs between men and women, likely due to anatomical and hormonal differences. Importantly, the urobiome has been associated with a variety of lower urinary tract disorders, including overactive bladder and post-operative urinary tract infection, raising the possibility that clinicians might one day treat symptoms by modifying the urobiome instead of killing the suspected uropathogen. Little is known concerning the relationship between the urobiome and host genetics; so far, only a single paper has reported such a study. However, major efforts have gone into understanding the genomics of the urobiome itself, a process facilitated by the fact that many urobiome studies have used metaculturomic methods to detect and identify microbes. In this narrative review, we will introduce the urobiome with separate sections on the female and male urobiomes, discuss challenges specific to the urobiome, describe newly discovered associations between the urobiome and lower urinary tract symptoms, and highlight the one study that has attempted to relate host genetics and the urobiome. We will finish with a section on how metagenomic surveys and whole genome sequencing of bacterial isolates are improving our understanding of the urobiome and its relationship to lower urinary tract health and disorders.

The urobiome of continent adult women: a cross-sectional study.

OBJECTIVE: To characterise the bladder microbiota of continent adult women. DESIGN: Cross-sectional study of adult women who contributed catheterised urine samples, completed validated symptom questionnaires, and provided demographic data. SETTING: US academic medical centre. POPULATION: Well-characterised continent adult women. METHODS: Participants contributed symptoms questionnaires, demographic data, and catheterised urine samples that were analysed by enhanced urine culture methodology and 16S rRNA gene sequencing. MAIN OUTCOME MEASURES: Associations between demographics and microbial community state structures (urotypes, defined by the dominant taxon of each specimen). RESULTS: The bladder microbiota (urobiome) of a control group of 224 continent women were characterised, demonstrating variability in terms of urotype. The most common urotype was Lactobacillus (19%), which did not differ with any demographic. In contrast, the Gardnerella (P < 0.001) and Escherichia (P = 0.005) urotypes were more common in younger and older women, respectively. CONCLUSIONS: For urobiome research, enhanced culture methods and/or DNA sequencing are the preferred techniques for bacterial detection. The interpretation of clinical tests, such as the standard urine culture, should incorporate the knowledge that some women have Gardnerella or Escherichia urotypes without evidence of any clinical disorder. Clinical care strategies should preserve or restore the beneficial effects of the native urobiome, as disruption of that microbial community could result in unintended vulnerability to uropathogen invasion or opportunistic pathogen overgrowth. Longitudinal studies of urobiome responses to therapies should be encouraged. TWEETABLE ABSTRACT: In continent adult women bladder microbiome composition differs by age, with relevance for clinical practice.

A combination of assays reveals biomass differences in biofilms formed by Escherichia coli mutants.

AIMS: The aim of this study was to develop an assay system that can quantify the amount of biomass in biofilms formed by different isogenic mutants of an Escherichia coli K-12 strain. METHODS AND RESULTS: The reported assay, which is based on the BacTiter-Glo assay from Promega, uses bioluminescence to detect the intracellular concentration of ATP, which correlates with viable bacterial cell numbers. The quantitative data obtained with this ATP assay were compared to those obtained with the conventional crystal violet assay. As a qualitative control, scanning electron microscopy was performed. CONCLUSIONS: The ATP assay, the crystal violet assay and scanning electron microscopy yielded similar results for six of the eight strains tested. For the remaining two strains, the images from the scanning electron microscopy confirmed the results from the ATP assay. SIGNIFICANCE AND IMPACT OF THE STUDY: The ATP assay, in combination with other quantitative and qualitative assays, will allow us to perform genetic studies on the regulatory network that underlies the early steps in E. coli biofilm formation.

Mechanisms, Detection, and Relevance of Protein Acetylation in Prokaryotes.

Posttranslational modification of a protein, either alone or in combination with other modifications, can control properties of that protein, such as enzymatic activity, localization, stability, or interactions with other molecules. N-ε-Lysine acetylation is one such modification that has gained attention in recent years, with a prevalence and significance that rival those of phosphorylation. This review will discuss the current state of the field in bacteria and some of the work in archaea, focusing on both mechanisms of N-ε-lysine acetylation and methods to identify, quantify, and characterize specific acetyllysines. Bacterial N-ε-lysine acetylation depends on both enzymatic and nonenzymatic mechanisms of acetylation, and recent work has shed light into the regulation of both mechanisms. Technological advances in mass spectrometry have allowed researchers to gain insight with greater biological context by both (i) analyzing samples either with stable isotope labeling workflows or using label-free protocols and (ii) determining the true extent of acetylation on a protein population through stoichiometry measurements. Identification of acetylated lysines through these methods has led to studies that probe the biological significance of acetylation. General and diverse approaches used to determine the effect of acetylation on a specific lysine will be covered.

Twist state phenotypes of Bacillus subtilis macrofibre mutants.

The range of macrofibre twist states that can be achieved by various strains of Bacillus subtilis has been examined as a function of two variables: growth temperature and medium composition. Two graphic techniques were utilized to organize and compare data which pertain to the complex phenotypes of macrofibre mutants. The steady state twist states of strains were determined by qualitative examination. Structures were produced at each of the extremes of temperature and medium composition. Patterns obtained from a graphical representation of these data permitted the strains to be grouped into three classes: (A) strains in which helix-hand inversion could be triggered by nutrition at both 20 degrees or 48 degrees C, and by temperature in either medium; (B) strains in which a more limited set of conditions could induce inversion, and (C) strains which were restricted to either the right- or left-hand domain of twist states. Genetic factors governing these patterns were examined. Quantitative measurements of static twist were obtained over the entire temperature and media range, providing a detailed picture of the dependence of twist upon these environmental influences. Although the macrofibre twist state phenotype (as a function of both variables over the entire range of conditions) of each strain was unique, common features were discernible in all strains. Although some strains were limited to a single helix hand under all conditions studied, none were found to be restricted to a single twist state.

The short form of the CheA protein restores kinase activity and chemotactic ability to kinase-deficient mutants.

Escherichia coli expresses two forms of the chemotaxis-associated CheA protein, CheAL and CheAS, as the result of translational initiation at two distinct, in-frame initiation sites in the gene cheA. The long form, CheAL, plays a crucial role in the chemotactic signal transduction mechanism by phosphorylating two other chemotaxis proteins: CheY and CheB. CheAL must first autophosphorylate at amino acid His-48 before transferring its phosphono group to these other signal transduction proteins. The short form, CheAS, lacks the N-terminal 97 amino acids of CheAL and, therefore, does not possess the site of autophosphorylation. Here we demonstrate that although it lacks the ability to autophosphorylate, CheAS can mediate phosphorylation of kinase-deficient variants of CheAL each of which retains a functional autophosphorylation site. This transphosphorylation enables these kinase-deficient CheAL variants to phosphorylate CheY. Because it mediates this activity, CheAS can restore to kinase-deficient E. coli cells the ability to tumble and, thus, to perform chemotaxis in swarm plate assays.

Migration of bacteria in semisolid agar.

We studied the migration through semisolid agar of chemotactic and nonchemotactic cells of Escherichia coli. While swarms of nonchemotactic cells were generally smaller than those of chemotactic cells, they varied markedly in size and in structure. Cells that failed to tumble or that tumbled incessantly formed the smallest swarms. Cells that tumbled at intermediate frequencies formed much larger swarms, even when deleted for many of the genes known to be required for chemotaxis. Surprisingly, the higher the tumble frequency, the larger the swarms. Microscopic examination revealed that tumbles enable cells to back away from obstructions in the agar. Thus, not all cells that swarm effectively need be chemotactic.

Regulation of acetyl phosphate synthesis and degradation, and the control of flagellar expression in Escherichia coli.

We investigated the relationship between Escherichia coli flagellar expression and the regulation of acetyl phosphate synthesis and degradation. Using cells either wild type for acetyl phosphate metabolism or defective for phosphotransacetylase or acetate kinase, or both, we measured flagellar expression and the intracellular concentration of acetyl phosphate relative to growth phase and temperature. Under the conditions tested, we found that elevated levels of acetyl phosphate corresponded to inhibition of flagellar synthesis. To extend these observations, we measured the intracellular concentration of acetyl-CoA, the level of expression from the pta and ackA promoters, and the activities of phosphotransacetylase and acetate kinase derived from cell lysates. Relative to increasing culture density, acetyl-CoA levels and expression from both the pta and ackA promoters decreased. Relative to increasing temperature, expression from the ackA promoter decreased and phosphotransacetylase activity increased. In contrast, temperature had little or no effect on either acetate kinase activity or expression from the pta promoter. We propose that cells regulate intracellular acetyl phosphate concentrations relative to growth phase and temperature by modulating the availability of acetyl-CoA, the expression of ackA, and the activity of phosphotransacetylase.

Characterization of nutrition-induced helix hand inversion of Bacillus subtilis macrofibers.

The kinetics of Bacillus subtilis macrofiber helix hand inversion was examined. Inversion was induced by transfer of structures produced in one medium to another medium. When cultured at 20 degrees C in either medium, the doubling time was approximately 100 min. To establish a baseline, the macrofiber twist state produced in one medium was measured over the same time course during which other macrofibers underwent inversion after transfer to a second medium. The baseline was used to identify the time of inversion initiation: the point at which curves representing changes of twist as a function of time after transfer to the new medium intersected the baseline. Right- and left-handed macrofibers of different twists were produced by growth in mixtures of TB and S1 media. These were used to determine the influence of initial twist on the time course of inversion initiation. In the right to left inversion, a positive correlation was found between initial twist and the time of inversion initiation. The left to right inversion differed, however, in that a constant time was required for inversion initiation regardless of the starting left-handed twist. When a nutritional pulse was administered by transferring fibers from TB to S1 to TB medium, the time to initiation of inversion was found to decrease with incubation of increasing duration in S1 medium. A similar pulse protocol was used in conjunction with inhibitors to examine the protein and peptidoglycan synthesis requirements for the establishment of nutrition-induced memory that leads to initiation of inversion. Nutritionally induced right to left inversion but not left to right inversion required protein synthesis. The addition of trypsin to left-handed macrofibers apparently required, as described previously for the temperature-regulated twist system (D. Favre, D. Karamata, and N. H. Mendelson, J. Bacteriol. 164:1141-1145, 1985), for the production of left-handed twist states in the nutrition system.

Coexpression of the long and short forms of CheA, the chemotaxis histidine kinase, by members of the family Enterobacteriaceae.

CheA is the histidine protein kinase of a two-component signal transduction system required for bacterial chemotaxis. Motile cells of the enteric species Escherichia coli and Salmonella typhimurium synthesize two forms of CheA by utilizing in-frame initiation sites within the gene cheA. The full-length protein, CheAL, plays an essential role in the chemotactic signaling pathway. In contrast, the function of the short form, CheAs, remains elusive. Although CheAs lacks the histidine residue that becomes phosphorylated in CheAL, it exhibits both kinase activity and the ability to interact with and enhance the activity of CheZ, a chemotaxis protein that accelerates dephosphorylation of the two-component response regulator CheY. To determine whether other members of the family Enterobacteriaceae express CheAs and CheZ, we analyzed immunoblots of proteins from clinical isolates of a variety of enteric species. All motile, chemotactic isolates that we tested coexpressed CheAL, CheAs, and CheZ. The only exceptions were closely related plant pathogens of the genus Erwinia, which expressed CheAL and CheZ but not CheAs. We also analyzed nucleotide sequences of the cheA loci from isolates of Serratia marcescens and Enterobacter cloacae, demonstrating the presence of in-frame translation initiation sites similar to those observed in the cheA loci of E. coli and S. typhimurium. Since coexpression of CheAs and CheZ appears to be limited to motile, chemotactic enteric bacteria, we propose that CheAs may play an important role in chemotactic responses in some environmental niches encountered by enteric species.

Acetyladenylate plays a role in controlling the direction of flagellar rotation.

Cells of Escherichia coli deleted for genes that code for the transducers and all the known cytoplasmic Che proteins except CheY responded reversibly to the addition of acetate by spinning their flagellar motors clockwise. By varying growth conditions and using metabolic inhibitors and mutants deficient in acetate metabolism, this effect was shown to require acetate-CoA synthetase [acetate:CoA ligase (AMP-forming); EC 6.2.1.1], an enzyme that catalyzes the formation of acetyl-CoA from acetate by an acetyladenylate intermediate. A mutant deficient in this enzyme but retaining the chemotaxis genes was deficient for chemotaxis. Thus, acetyladenylate appears to play a role in generating clockwise rotation at the level of CheY or the motor.

Regulation of Bacillus subtilis macrofiber twist development by D-alanine.

Twist states of Bacillus subtilis macrofibers were found to vary as a function of the concentration of D-alanine in the medium during growth. L-Alanine in the same concentration range had no effect. Increasing concentrations of D-alanine resulted in structures progressively more right-handed (or less left-handed). All strains examined in this study, including mutants fixed in the left-hand domain as a function of temperature, responded to D-alanine in the same way. All twist states from tight left- to tight right-handedness could be achieved solely by varying the D-alanine concentration. The D-alanine-requiring macrofiber strain 2C8, which carries a genetic defect (dal-1) in the alanine racemase, behaved in a similar fashion. The combined effects of D-alanine and ammonium sulfate (a factor known to influence macrofiber twist development in the leftward direction) were examined by using both strains able to undergo temperature-induced helix hand inversion and others incapable of doing so. In all cases, the effects of D-alanine predominated. A synergism was found in which increasing the concentration of ammonium sulfate in the presence of D-alanine enhanced the right-factor activity of the latter. A D-alanine pulse protocol provided evidence that structures undergo a transient inversion indicative of "memory." Chloramphenicol treatment inhibited the establishment of memory in the D-alanine-induced right to left inversion, supporting the existence of a "left twist protein(s)" that is required for the attainment of left-handed twist states. Chemical analysis of cell walls obtained from right- and left-handed macrofibers produced in the presence and absence of D-alanine, respectively, failed to reveal twist state-specific differences in the overall composition of either peptidoglycan or wall teichoic acids.

The short form of CheA couples chemoreception to CheA phosphorylation.

Escherichia coli cells express two forms of the chemotaxis-associated CheA protein, CheAL and CheAS, as the result of translational initiation at two distinct in-frame initiation sites in the gene cheA. The long form, CheAL, plays a crucial role in chemotactic signal transduction. As a histidine protein kinase, it first autophosphorylates at amino acid His-48; then, it phosphorylates two other chemotaxis proteins, CheY and CheB. The short form, CheAS, lacks the amino-terminal 97 amino acids of CheAL and, therefore, does not contain the site of autophosphorylation. However, it does retain a functional kinase domain. As a consequence, CheAS can mediate transphosphorylation of kinase-deficient CheAL variants. Here we demonstrate in vitro that CheAS also can mediate transphosphorylation of a CheAL variant that lacks the C-terminal segment, a portion of the protein which is thought to interact with CheW and the chemoreceptors. The presence of CheW and the chemoreceptor Tsr enhances this activity and results in modulation of the transphosphorylation rate in response to the Tsr ligand, L-serine. Because CheAS can mediate this activity, it can restore chemotactic ability to Escherichia coli cells that express this truncated CheAL variant.

Genetic analysis of the catalytic domain of the chemotaxis-associated histidine kinase CheA.

Escherichia coli cells express two forms of CheA, the histidine kinase associated with chemotaxis. The long form, CheA(L), plays a critical role in chemotactic signal transduction by phosphorylating two chemotaxis-associated response regulators, CheY and CheB. CheA(L) first autophosphorylates amino acid His-48 before its phosphoryl group is transferred to these response regulators. The short form, CheA(S), lacks the amino-terminal 97 amino acids of CheA(L) and therefore does not possess the site of phosphorylation. The centrally located transmitter domain of both forms of CheA contains four regions, called N, G1, F, and G2, highly conserved among histidine kinases of the family of two-component signal transduction systems. On the basis of sequence similarity to highly conserved regions of certain eukaryotic kinases, the G1 and G2 regions are purported to be involved in the binding and hydrolysis of ATP. We report here that alleles mutated in the G1, G2, or F region synthesize CheA variants that cannot autophosphorylate in vitro and which cannot support chemotaxis in vivo. We also show that in vitro, the nonphosphorylatable CheA(S) protein mediates transphosphorylation of a CheA(L) variant defective in both G1 and G2. In contrast, CheA(L) variants defective for either G1 or G2 mediate transphosphorylation of each other poorly, if at all. These results are consistent with a mechanism by which the G1 and G2 regions of one protomer of a CheA dimer form a unit that mediates transphosphorylation of the other protomer within that dimer.

Genetic analysis of the nuo locus, which encodes the proton-translocating NADH dehydrogenase in Escherichia coli.

Complex I (EC 1.6.99.3) of the bacterium Escherichia coli is considered to be the minimal form of the type I NADH dehydrogenase, the first enzyme complex in the respiratory chain. Because of its small size and relative simplicity, the E. coli enzyme has become a model used to identify and characterize the mechanism(s) by which cells regulate the synthesis and assembly of this large respiratory complex. To begin dissecting the processes by which E. coli cells regulate the expression of nuo and the assembly of complex I, we undertook a genetic analysis of the nuo locus, which encodes the 14 Nuo subunits comprising E. coli complex I. Here we present the results of studies, performed on an isogenic collection of nuo mutants, that focus on the physiological, biochemical, and molecular consequences caused by the lack of or defects in several Nuo subunits. In particular, we present evidence that NuoG, a peripheral subunit, is essential for complex I function and that it plays a role in the regulation of nuo expression and/or the assembly of complex I.

sigma(70) is the principal sigma factor responsible for transcription of acs, which encodes acetyl coenzyme A synthetase in Escherichia coli.

Cells of Escherichia coli undergo a metabolic switch associated with the production and utilization of acetate. During exponential growth on tryptone broth, these cells excrete acetate via the phosphotransacetylase-acetate kinase (Pta-AckA) pathway. As they begin the transition to stationary phase, they instead resorb acetate, activate it to acetyl coenzyme A (acetyl-CoA) by means of the enzyme acetyl-CoA synthetase (Acs) and utilize it to generate energy and biosynthetic components via the tricarboxylic acid cycle and the glyoxylate shunt, respectively. This metabolic switch depends upon the induction of Acs. As part of our effort to dissect the mechanism(s) underlying induction and to identify the signal(s) that triggers that induction, we sought the sigma factor most responsible for acs expression. Using isogenic strains that carry a temperature sensitivity allele of the gene that encodes sigma(70) and either a wild-type or null allele of the gene that encodes sigma(S), we determined by immunoblotting, reverse transcriptase PCR, and acs::lacZ transcriptional fusion analyses that sigma(70) is the sigma factor primarily responsible for the acs transcription that cells induce during mid-exponential phase. In contrast, sigma(S) partially inhibits that transcription as cells enter stationary phase.

Cloning, characterization, and functional expression of acs, the gene which encodes acetyl coenzyme A synthetase in Escherichia coli.

Acetyl coenzyme A synthetase (Acs) activates acetate to acetyl coenzyme A through an acetyladenylate intermediate; two other enzymes, acetate kinase (Ack) and phosphotransacetylase (Pta), activate acetate through an acetyl phosphate intermediate. We subcloned acs, the Escherichia coli open reading frame purported to encode Acs (F. R. Blattner, V. Burland, G. Plunkett III, H. J. Sofia, and D. L. Daniels, Nucleic Acids Res. 21:5408-5417, 1993). We constructed a mutant allele, delta acs::Km, with the central 0.72-kb BclI-BclI portion of acs deleted, and recombined it into the chromosome. Whereas wild-type cells grew well on acetate across a wide range of concentrations (2.5 to 50 mM), those deleted for acs grew poorly on low concentrations (< or = 10 mM), those deleted for ackA and pta (which encode Ack and Pta, respectively) grew poorly on high concentrations (> or = 25 mM), and those deleted for acs, ackA, and pta did not grow on acetate at any concentration tested. Expression of acs from a multicopy plasmid restored growth to cells deleted for all three genes. Relative to wild-type cells, those deleted for acs did not activate acetate as well, those deleted for ackA and pta displayed even less activity, and those deleted for all three genes did not activate acetate at any concentration tested. Induction of acs resulted in expression of a 72-kDa protein, as predicted by the reported sequence. This protein immunoreacted with antiserum raised against purified Acs isolated from an unrelated species, Methanothrix soehngenii. The purified E. coli Acs then was used to raise anti-E. coli Acs antiserum, which immunoreacted with a 72-kDa protein expressed by wild-type cells but not by those deleted for acs. When purified in the presence, but not in the absence, of coenzyme A, the E. coli enzyme activated acetate across a wide range of concentrations in a coenzyme A-dependent manner. On the basis of these and other observations, we conclude that this open reading frame encodes the acetate-activating enzyme, Acs.

Both CheA and CheW are required for reconstitution of chemotactic signaling in Escherichia coli.

If cells of Escherichia coli deleted for genes that specify transducers and all known cytoplasmic chemotaxis proteins are reconstituted with CheA, CheW, and CheY, they spin their flagella alternately clockwise and counterclockwise. If the aspartate receptor also is present, clockwise rotation is suppressed upon addition of aspartate. If either CheA or CheW is absent, the fraction of time that the flagella spin clockwise is reduced and responses to aspartate do not occur.