scholarly journals Global Transcriptional Regulators Fine-Tune the Translational and Metabolic Efficiency for Optimal Growth of Escherichia coli

mSystems ◽  
2021 ◽  
Vol 6 (2) ◽  
Author(s):  
Mahesh S. Iyer ◽  
Ankita Pal ◽  
Sumana Srinivasan ◽  
Pramod R. Somvanshi ◽  
K. V. Venkatesh
Keyword(s):  
Escherichia Coli ◽  
Exponential Growth ◽  
Molecular Mechanisms ◽  
Transcriptional Regulatory Network ◽  
Expression Profiles ◽  
Transcriptional Regulators ◽  
Metabolic Efficiency ◽  
Metabolic Processes ◽  
Intracellular Metabolites ◽  
Content Type

ABSTRACT Global transcriptional regulators coordinate complex genetic interactions that bestow better adaptability for an organism against external and internal perturbations. These transcriptional regulators are known to control an enormous array of genes with diverse functionalities. However, regulator-driven molecular mechanisms that underpin precisely tuned translational and metabolic processes conducive for rapid exponential growth remain obscure. Here, we comprehensively reveal the fundamental role of global transcriptional regulators FNR, ArcA, and IHF in sustaining translational and metabolic efficiency under glucose fermentative conditions in Escherichia coli. By integrating high-throughput gene expression profiles and absolute intracellular metabolite concentrations, we illustrate that these regulators are crucial in maintaining nitrogen homeostasis, govern expression of otherwise unnecessary or hedging genes, and exert tight control on metabolic bottleneck steps. Furthermore, we characterize changes in expression and activity profiles of other coregulators associated with these dysregulated metabolic pathways, determining the regulatory interactions within the transcriptional regulatory network. Such systematic findings emphasize their importance in optimizing the proteome allocation toward metabolic enzymes as well as ribosomes, facilitating condition-specific phenotypic outcomes. Consequentially, we reveal that disruption of this inherent trade-off between ribosome and metabolic proteome economy due to the loss of regulators resulted in lowered growth rates. Moreover, our findings reinforce that the accumulations of intracellular metabolites in the event of proteome repartitions negatively affects the glucose uptake. Overall, by extending the three-partition proteome allocation theory concordant with multi-omics measurements, we elucidate the physiological consequences of loss of global regulators on central carbon metabolism restraining the organism to attain maximal biomass synthesis. IMPORTANCE Cellular proteome allocation in response to environmental or internal perturbations governs their eventual phenotypic outcome. This entails striking an effective balance between amino acid biosynthesis by metabolic proteins and its consumption by ribosomes. However, the global transcriptional regulator-driven molecular mechanisms that underpin their coordination remains unexplored. Here, we emphasize that global transcriptional regulators, known to control expression of a myriad of genes, are fundamental for precisely tuning the translational and metabolic efficiencies that define the growth optimality. Towards this, we systematically characterized the single deletion effect of FNR, ArcA, and IHF regulators of Escherichia coli on exponential growth under anaerobic glucose fermentative conditions. Their absence disrupts the stringency of proteome allocation, which manifests as impairment in key metabolic processes and an accumulation of intracellular metabolites. Furthermore, by incorporating an extension to the empirical growth laws, we quantitatively demonstrate the general design principles underlying the existence of these regulators in E. coli.

scholarly journals Physiological Evidence for Isopotential Tunneling in the Electron Transport Chain of Methane-Producing Archaea

2017 ◽  
Vol 83 (18) ◽  
Author(s):  
Nikolas Duszenko ◽  
Nicole R. Buan
Keyword(s):  
Escherichia Coli ◽  
Electron Transport ◽  
Stationary Phase ◽  
Exponential Growth ◽  
Electron Transport Chain ◽  
Methanosarcina Barkeri ◽  
Metabolic Efficiency ◽  
Methanosarcina Acetivorans ◽  
Content Type ◽  
Transport Chain

ABSTRACT Many, but not all, organisms use quinones to conserve energy in their electron transport chains. Fermentative bacteria and methane-producing archaea (methanogens) do not produce quinones but have devised other ways to generate ATP. Methanophenazine (MPh) is a unique membrane electron carrier found in Methanosarcina species that plays the same role as quinones in the electron transport chain. To extend the analogy between quinones and MPh, we compared the MPh pool sizes between two well-studied Methanosarcina species, Methanosarcina acetivorans C2A and Methanosarcina barkeri Fusaro, to the quinone pool size in the bacterium Escherichia coli. We found the quantity of MPh per cell increases as cultures transition from exponential growth to stationary phase, and absolute quantities of MPh were 3-fold higher in M. acetivorans than in M. barkeri. The concentration of MPh suggests the cell membrane of M. acetivorans, but not of M. barkeri, is electrically quantized as if it were a single conductive metal sheet and near optimal for rate of electron transport. Similarly, stationary (but not exponentially growing) E. coli cells also have electrically quantized membranes on the basis of quinone content. Consistent with our hypothesis, we demonstrated that the exogenous addition of phenazine increases the growth rate of M. barkeri three times that of M. acetivorans. Our work suggests electron flux through MPh is naturally higher in M. acetivorans than in M. barkeri and that hydrogen cycling is less efficient at conserving energy than scalar proton translocation using MPh. IMPORTANCE Can we grow more from less? The ability to optimize and manipulate metabolic efficiency in cells is the difference between commercially viable and nonviable renewable technologies. Much can be learned from methane-producing archaea (methanogens) which evolved a successful metabolic lifestyle under extreme thermodynamic constraints. Methanogens use highly efficient electron transport systems and supramolecular complexes to optimize electron and carbon flow to control biomass synthesis and the production of methane. Worldwide, methanogens are used to generate renewable methane for heat, electricity, and transportation. Our observations suggest Methanosarcina acetivorans, but not Methanosarcina barkeri, has electrically quantized membranes. Escherichia coli, a model facultative anaerobe, has optimal electron transport at the stationary phase but not during exponential growth. This study also suggests the metabolic efficiency of bacteria and archaea can be improved using exogenously supplied lipophilic electron carriers. The enhancement of methanogen electron transport through methanophenazine has the potential to increase renewable methane production at an industrial scale.

scholarly journals Global transcriptional regulators fine-tune the translational and metabolic machinery in Escherichia coli under anaerobic fermentation

2020 ◽  
Author(s):  
Mahesh S. Iyer ◽  
Ankita Pal ◽  
Sumana Srinivasan ◽  
Pramod R. Somvanshi ◽  
K.V. Venkatesh
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Exponential Growth ◽  
Metabolic Networks ◽  
Anaerobic Fermentation ◽  
Cost Effective ◽  
Optimal Growth ◽  
Transcriptional Regulators ◽  
Metabolic Efficiency ◽  
Global Regulators

AbstractComplex regulatory interactions between genetic and metabolic networks together confer robustness against external and internal perturbations in an organism such as Escherichia coli. In balanced exponential growth, this robustness is attributed to cost-effective metabolism by means of efficient resource allocation coordinated by the interplay of global transcriptional regulators with growth-rate dependent machinery. Here, we reappraise the role of global transcriptional regulators FNR, ArcA and IHF, integral to sustaining proteome-efficiency in anaerobic fermentative conditions, fundamental for optimal growth of E. coli. We reveal at the transcriptome and metabolome level, that absence of these global regulators ensued a disruption of nitrogen homeostasis, overexpression of otherwise unnecessary or hedging genes and impairment in core bottleneck steps and amino acid metabolism. Notably, our findings emphasize their importance in optimizing the metabolic proteome resources essential for rapid exponential growth. Consequentially, the perturbations in the metabolic proteome as a result of deletion of global regulators unbalances the ribosomal proteome share imposing a high translation program, though at the expense of lowered efficiency. We illustrate that disruption of this inherent trade-off between metabolic and ribosomal proteomic investment eventually culminate to lowered growth rates. Despite no changes in gene expression related to glucose import, our findings elucidate that the accumulations of intracellular metabolites directly modulated by growth rate, negatively impacts the glucose uptake. Our results employing the proteome allocation theory and quantitative experimental measurements, suffices to explain the physiological consequences of altered translational and metabolic efficiency in the cell, driven by the loss of these global regulators.

scholarly journals Biophysical Properties of Escherichia coli Cytoplasm in Stationary Phase by Superresolution Fluorescence Microscopy

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Yanyu Zhu ◽  
Mainak Mustafi ◽  
James C. Weisshaar
Keyword(s):  
Escherichia Coli ◽  
Fluorescence Microscopy ◽  
Rna Polymerase ◽  
Stationary Phase ◽  
Exponential Growth ◽  
Mean Square Displacement ◽  
Quiescent State ◽  
Chromosomal Dna ◽  
Content Type ◽  
E Coli

ABSTRACT In nature, bacteria must survive long periods of nutrient deprivation while maintaining the ability to recover and grow when conditions improve. This quiescent state is called stationary phase. The biochemistry of Escherichia coli in stationary phase is reasonably well understood. Much less is known about the biophysical state of the cytoplasm. Earlier studies of harvested nucleoids concluded that the stationary-phase nucleoid is “compacted” or “supercompacted,” and there are suggestions that the cytoplasm is “glass-like.” Nevertheless, stationary-phase bacteria support active transcription and translation. Here, we present results of a quantitative superresolution fluorescence study comparing the spatial distributions and diffusive properties of key components of the transcription-translation machinery in intact E. coli cells that were either maintained in 2-day stationary phase or undergoing moderately fast exponential growth. Stationary-phase cells are shorter and exhibit strong heterogeneity in cell length, nucleoid volume, and biopolymer diffusive properties. As in exponential growth, the nucleoid and ribosomes are strongly segregated. The chromosomal DNA is locally more rigid in stationary phase. The population-weighted average of diffusion coefficients estimated from mean-square displacement plots is 2-fold higher in stationary phase for both RNA polymerase (RNAP) and ribosomal species. The average DNA density is roughly twice as high as that in cells undergoing slow exponential growth. The data indicate that the stationary-phase nucleoid is permeable to RNAP and suggest that it is permeable to ribosomal subunits. There appears to be no need to postulate migration of actively transcribed genes to the nucleoid periphery. IMPORTANCE Bacteria in nature usually lack sufficient nutrients to enable growth and replication. Such starved bacteria adapt into a quiescent state known as the stationary phase. The chromosomal DNA is protected against oxidative damage, and ribosomes are stored in a dimeric structure impervious to digestion. Stationary-phase bacteria can recover and grow quickly when better nutrient conditions arise. The biochemistry of stationary-phase E. coli is reasonably well understood. Here, we present results from a study of the biophysical state of starved E. coli. Superresolution fluorescence microscopy enables high-resolution location and tracking of a DNA locus and of single copies of RNA polymerase (the transcription machine) and ribosomes (the translation machine) in intact E. coli cells maintained in stationary phase. Evidently, the chromosomal DNA remains sufficiently permeable to enable transcription and translation to occur. This description contrasts with the usual picture of a rigid stationary-phase cytoplasm with highly condensed DNA.

scholarly journals The Escherichia coli transcriptome mostly consists of independently regulated modules

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Anand V. Sastry ◽  
Ye Gao ◽  
Richard Szubin ◽  
Ying Hefner ◽  
Sibei Xu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Transcriptional Regulatory Network ◽  
Transcriptional Regulators ◽  
Specific Gene ◽  
E Coli ◽  
Gene Sets ◽  
Transcriptional Regulatory ◽  
Genetic Perturbations ◽  
Function Discovery ◽  
Multiple Strains

AbstractUnderlying cellular responses is a transcriptional regulatory network (TRN) that modulates gene expression. A useful description of the TRN would decompose the transcriptome into targeted effects of individual transcriptional regulators. Here, we apply unsupervised machine learning to a diverse compendium of over 250 high-quality Escherichia coli RNA-seq datasets to identify 92 statistically independent signals that modulate the expression of specific gene sets. We show that 61 of these transcriptomic signals represent the effects of currently characterized transcriptional regulators. Condition-specific activation of signals is validated by exposure of E. coli to new environmental conditions. The resulting decomposition of the transcriptome provides: a mechanistic, systems-level, network-based explanation of responses to environmental and genetic perturbations; a guide to gene and regulator function discovery; and a basis for characterizing transcriptomic differences in multiple strains. Taken together, our results show that signal summation describes the composition of a model prokaryotic transcriptome.

scholarly journals Altered Regulation of the Diguanylate Cyclase YaiC Reduces Production of Type 1 Fimbriae in a Pst Mutant of Uropathogenic Escherichia coli CFT073

2017 ◽  
Vol 199 (24) ◽  
Author(s):  
Sébastien Crépin ◽  
Gaëlle Porcheron ◽  
Sébastien Houle ◽  
Josée Harel ◽  
Charles M. Dozois
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Molecular Mechanisms ◽  
Diguanylate Cyclase ◽  
Altered Expression ◽  
Content Type ◽  
Type 1 Fimbriae ◽  
Tract Infections ◽  
Pst System

ABSTRACT The pst gene cluster encodes the phosphate-specific transport (Pst) system. Inactivation of the Pst system constitutively activates the two-component regulatory system PhoBR and attenuates the virulence of pathogenic bacteria. In uropathogenic Escherichia coli strain CFT073, attenuation by inactivation of pst is predominantly attributed to the decreased expression of type 1 fimbriae. However, the molecular mechanisms connecting the Pst system and type 1 fimbriae are unknown. To address this, a transposon library was constructed in the pst mutant, and clones were tested for a regain in type 1 fimbrial production. Among them, the diguanylate cyclase encoded by yaiC (adrA in Salmonella) was identified to connect the Pst system and type 1 fimbrial expression. In the pst mutant, the decreased expression of type 1 fimbriae is connected by the induction of yaiC. This is predominantly due to altered expression of the FimBE-like recombinase genes ipuA and ipbA, affecting at the same time the inversion of the fim promoter switch (fimS). In the pst mutant, inactivation of yaiC restored fim-dependent adhesion to bladder cells and virulence. Interestingly, the expression of yaiC was activated by PhoB, since transcription of yaiC was linked to the PhoB-dependent phoA-psiF operon. As YaiC is involved in cyclic di-GMP (c-di-GMP) biosynthesis, an increased accumulation of c-di-GMP was observed in the pst mutant. Hence, the results suggest that one mechanism by which deletion of the Pst system reduces the expression of type 1 fimbriae is through PhoBR-mediated activation of yaiC, which in turn increases the accumulation of c-di-GMP, represses the fim operon, and, consequently, attenuates virulence in the mouse urinary tract infection model. IMPORTANCE Urinary tract infections (UTIs) are common bacterial infections in humans. They are mainly caused by uropathogenic Escherichia coli (UPEC). We previously showed that interference with phosphate homeostasis decreases the expression of type 1 fimbriae and attenuates UPEC virulence. Herein, we identified that alteration of the phosphate metabolism increases production of the signaling molecule c-di-GMP, which in turn decreases the expression of type 1 fimbriae. We also determine the regulatory cascade leading to the accumulation of c-di-GMP and identify the Pho regulon as new players in c-di-GMP-mediated cell signaling. By understanding the molecular mechanisms leading to the expression of virulence factors, we will be in a better position to develop new therapeutics.

scholarly journals In Vivo mRNA Profiling of Uropathogenic Escherichia coli from Diverse Phylogroups Reveals Common and Group-Specific Gene Expression Profiles

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Piotr Bielecki ◽  
Uthayakumar Muthukumarasamy ◽  
Denitsa Eckweiler ◽  
Agata Bielecka ◽  
Sarah Pohl ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Specific Gene ◽  
Content Type ◽  
E Coli ◽  
Human Host ◽  
Tract Infections

ABSTRACTmRNA profiling of pathogens during the course of human infections gives detailed information on the expression levels of relevant genes that drive pathogenicity and adaptation and at the same time allows for the delineation of phylogenetic relatedness of pathogens that cause specific diseases. In this study, we used mRNA sequencing to acquire information on the expression ofEscherichia colipathogenicity genes during urinary tract infections (UTI) in humans and to assign the UTI-associatedE. coliisolates to different phylogenetic groups. Whereas thein vivogene expression profiles of the majority of genes were conserved among 21E. colistrains in the urine of elderly patients suffering from an acute UTI, the specific gene expression profiles of the flexible genomes was diverse and reflected phylogenetic relationships. Furthermore, genes transcribedin vivorelative to laboratory media included well-described virulence factors, small regulatory RNAs, as well as genes not previously linked to bacterial virulence. Knowledge on relevant transcriptional responses that drive pathogenicity and adaptation of isolates to the human host might lead to the introduction of a virulence typing strategy into clinical microbiology, potentially facilitating management and prevention of the disease.IMPORTANCEUrinary tract infections (UTI) are very common; at least half of all women experience UTI, most of which are caused by pathogenicEscherichia colistrains. In this study, we applied massive parallel cDNA sequencing (RNA-seq) to provide unbiased, deep, and accurate insight into the nature and the dimension of the uropathogenicE. coligene expression profile during an acute UTI within the human host. This work was undertaken to identify key players in physiological adaptation processes and, hence, potential targets for new infection prevention and therapy interventions specifically aimed at sabotaging bacterial adaptation to the human host.

scholarly journals Characterization of Multidrug-Resistant Escherichia coli Isolates from Animals Presenting at a University Veterinary Hospital

2011 ◽  
Vol 77 (20) ◽  
pp. 7104-7112 ◽  
Author(s):  
Maria Karczmarczyk ◽  
Yvonne Abbott ◽  
Ciara Walsh ◽  
Nola Leonard ◽  
Séamus Fanning
Keyword(s):  
Escherichia Coli ◽  
Molecular Mechanisms ◽  
Gene Array ◽  
Multidrug Resistant ◽  
Genetic Determinants ◽  
Gene Encoding ◽  
Content Type ◽  
E Coli ◽  
Class 1

ABSTRACTIn this study, we examined molecular mechanisms associated with multidrug resistance (MDR) in a collection ofEscherichia coliisolates recovered from hospitalized animals in Ireland. PCR and DNA sequencing were used to identify genes associated with resistance. Class 1 integrons were prevalent (94.6%) and contained gene cassettes recognized previously and implicated mainly in resistance to aminoglycosides, β-lactams, and trimethoprim (aadA1,dfrA1-aadA1,dfrA17-aadA5,dfrA12-orfF-aadA2,blaOXA-30-aadA1,aacC1-orf1-orf2-aadA1,dfr7). Class 2 integrons (13.5%) contained thedfrA1-sat1-aadA1gene array. The most frequently occurring phenotypes included resistance to ampicillin (97.3%), chloramphenicol (75.4%), florfenicol (40.5%), gentamicin (54%), neomycin (43.2%), streptomycin (97.3%), sulfonamide (98.6%), and tetracycline (100%). The associated resistance determinants detected includedblaTEM,cat,floR,aadB,aphA1,strA-strB,sul2, andtet(B), respectively. TheblaCTX-M-2gene, encoding an extended-spectrum β-lactamase (ESβL), andblaCMY-2, encoding an AmpC-like enzyme, were identified in 8 and 18 isolates, respectively. The mobility of the resistance genes was demonstrated using conjugation assays with a representative selection of isolates. High-molecular-weight plasmids were found to be responsible for resistance to multiple antimicrobial compounds. The study demonstrated that animal-associated commensalE. coliisolates possess a diverse repertoire of transferable genetic determinants. Emergence of ESβLs and AmpC-like enzymes is particularly significant. To our knowledge, theblaCTX-M-2gene has not previously been reported in Ireland.

scholarly journals Transcriptional Responses of Escherichia coli K-12 and O157:H7 Associated with Lettuce Leaves

2012 ◽  
Vol 78 (6) ◽  
pp. 1752-1764 ◽  
Author(s):  
Ryan C. Fink ◽  
Elaine P. Black ◽  
Zhe Hou ◽  
Masayuki Sugawara ◽  
Michael J. Sadowsky ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Mechanisms ◽  
Leaf Surface ◽  
Short Term ◽  
Transcriptional Responses ◽  
Content Type ◽  
E Coli ◽  
Leaf Surfaces ◽  
K 12

ABSTRACTAn increasing number of outbreaks of gastroenteritis recently caused byEscherichia coliO157:H7 have been linked to the consumption of leafy green vegetables. Although it is known thatE. colisurvives and grows in the phyllosphere of lettuce plants, the molecular mechanisms by which this bacterium associates with plants are largely unknown. The goal of this study was to identifyE. coligenes relevant to its interaction, survival, or attachment to lettuce leaf surfaces, comparingE. coliK-12, a model system, andE. coliO157:H7, a pathogen associated with a large number of outbreaks. Using microarrays, we found that upon interaction with intact leaves, 10.1% and 8.7% of the 3,798 shared genes were differentially expressed in K-12 and O157:H7, respectively, whereas 3.1% changed transcript levels in both. The largest group of genes downregulated consisted of those involved in energy metabolism, includingtnaA(33-fold change), encoding a tryptophanase that converts tryptophan into indole. Genes involved in biofilm modulation (bhsAandybiM) and curli production (csgAandcsgB) were significantly upregulated inE. coliK-12 and O157:H7. BothcsgAandbhsA(ycfR) mutants were impaired in the long-term colonization of the leaf surface, but onlycsgAmutants had diminished ability in short-term attachment experiments. Our data suggested that the interaction ofE. coliK-12 and O157:H7 with undamaged lettuce leaves likely is initiated via attachment to the leaf surface using curli fibers, a downward shift in their metabolism, and the suppression of biofilm formation.

scholarly journals Survival of Escherichia coli Cells on Solid Copper Surfaces Is Increased by Glutathione

2014 ◽  
Vol 80 (22) ◽  
pp. 7071-7078 ◽  
Author(s):  
Cornelia Große ◽  
Grit Schleuder ◽  
Christin Schmole ◽  
Dietrich H. Nies
Keyword(s):  
Escherichia Coli ◽  
Molecular Mechanisms ◽  
Copper Tolerance ◽  
Anaerobic Growth ◽  
Resistant Bacteria ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Content Type ◽  
Copper Surfaces ◽  
Dps Protein ◽  
Solid Copper

ABSTRACTBacteria are rapidly killed on solid copper surfaces, so this material could be useful to limit the spread of multiple-drug-resistant bacteria in hospitals. InEscherichia coli, the DNA-protecting Dps protein and the NADH:ubiquinone oxidoreductase II Ndh were not involved in tolerance to copper ions or survival on solid copper surfaces. Decreased copper tolerance under anaerobic growth conditions in the presence of ascorbate and with melibiose as the carbon source indicated that sodium-dependent symport systems may provide an import route for CuIinto the cytoplasm. Glutathione-free ΔcopAΔgshAdouble mutants ofE. coliwere more rapidly inactivated on solid copper surfaces than glutathione-containing wild-type cells. Therefore, while DNA protection by Dps was not required, glutathione was needed to protect the cytoplasm and the DNA against damage mediated by solid copper surfaces, which may explain the differences in the molecular mechanisms of killing between glutathione-containing Gram-negative and glutathione-free Gram-positive bacteria.

scholarly journals 16S rRNA Gene Mutations Associated with Decreased Susceptibility to Tetracycline in Mycoplasma bovis

2014 ◽  
Vol 59 (2) ◽  
pp. 796-802 ◽  
Author(s):  
E. Amram ◽  
I. Mikula ◽  
C. Schnee ◽  
R. D. Ayling ◽  
R. A. J. Nicholas ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
16S Rrna ◽  
Molecular Mechanisms ◽  
Gene Mutations ◽  
Binding Pocket ◽  
Clinical Isolates ◽  
Mycoplasma Bovis ◽  
Rrna Gene ◽  
Content Type ◽  
Encoding Genes

ABSTRACTMycoplasma bovisisolates with decreased susceptibilities to tetracyclines are increasingly reported worldwide. The acquired molecular mechanisms associated with this phenomenon were investigated in 70 clinical isolates ofM. bovis. Sequence analysis of the two 16S rRNA-encoding genes (rrs3andrrs4alleles) containing the primary binding pocket for tetracycline (Tet-1 site) was performed on isolates with tetracycline hydrochloride MICs of 0.125 to 16 μg/ml. Mutations at positions A965T, A967T/C (Escherichia colinumbering) of helix 31, U1199C of helix 34, and G1058A/C were identified. Decreased susceptibilities to tetracycline (MICs, ≥2 μg/ml) were associated with mutations present at two (A965 and A967) or three positions (A965, A967, and G1058) of the tworrsalleles. Notet(M),tet(O), ortet(L) determinants were found in the genome of any of the 70M. bovisisolates. The data presented correlate (P< 0.0001) the mutations identified in the Tet-1 site of clinical isolates ofM. boviswith decreased susceptibility to tetracycline.

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