rpsU Gene Research Articles

Ribosomal mutations enable a switch between high fitness and high stress resistance in Listeria monocytogenes.

Multiple stress resistant variants of Listeria monocytogenes with mutations in rpsU encoding ribosomal protein RpsU have previously been isolated after a single exposure to acid stress. These variants, including L. monocytogenes LO28 variant V14 with a complete deletion of the rpsU gene, showed upregulation of the general stress sigma factor Sigma B-mediated stress resistance genes and had a lower maximum specific growth rate than the LO28 WT, signifying a trade-off between stress resistance and fitness. In the current work V14 has been subjected to an experimental evolution regime, selecting for higher fitness in two parallel evolving cultures. This resulted in two evolved variants with WT-like fitness: 14EV1 and 14EV2. Comparative analysis of growth performance, acid and heat stress resistance, in combination with proteomics and RNA-sequencing, indicated that in both lines reversion to WT-like fitness also resulted in WT-like stress sensitivity, due to lack of Sigma B-activated stress defense. Notably, genotyping of 14EV1 and 14EV2 provided evidence for unique point-mutations in the ribosomal rpsB gene causing amino acid substitutions at the same position in RpsB, resulting in RpsB22Arg-His and RpsB22Arg-Ser, respectively. Combined with data obtained with constructed RpsB22Arg-His and RpsB22Arg-Ser mutants in the V14 background, we provide evidence that loss of function of RpsU resulting in the multiple stress resistant and reduced fitness phenotype, can be reversed by single point mutations in rpsB leading to arginine substitutions in RpsB at position 22 into histidine or serine, resulting in a WT-like high fitness and low stress resistance phenotype. This demonstrates the impact of genetic changes in L. monocytogenes' ribosomes on fitness and stress resistance.

A single point mutation in the Listeria monocytogenes ribosomal gene rpsU enables SigB activation independently of the stressosome and the anti-sigma factor antagonist RsbV.

Microbial population heterogeneity leads to different stress responses and growth behavior of individual cells in a population. Previously, a point mutation in the rpsU gene (rpsUG50C) encoding ribosomal protein S21 was identified in a Listeria monocytogenes LO28 variant, which leads to increased multi-stress resistance and a reduced maximum specific growth rate. However, the underlying mechanisms of these phenotypic changes remain unknown. In L. monocytogenes, the alternative sigma factor SigB regulates the general stress response, with its activation controlled by a series of Rsb proteins, including RsbR1 and anti-sigma factor RsbW and its antagonist RsbV. We combined a phenotype and proteomics approach to investigate the acid and heat stress resistance, growth rate, and SigB activation of L. monocytogenes EGDe wild type and the ΔsigB, ΔrsbV, and ΔrsbR1 mutant strains. While the introduction of rpsUG50C in the ΔsigB mutant did not induce a SigB-mediated increase in robustness, the presence of rpsUG50C in the ΔrsbV and the ΔrsbR1 mutants led to SigB activation and concomitant increased robustness, indicating an alternative signaling pathway for the SigB activation in rpsUG50C mutants. Interestingly, all these rpsUG50C mutants exhibited reduced maximum specific growth rates, independent of SigB activation, possibly attributed to compromised ribosomal functioning. In summary, the increased stress resistance in the L. monocytogenes EGDe rpsUG50C mutant results from SigB activation through an unknown mechanism distinct from the classical stressosome and RsbV/RsbW partner switching model. Moreover, the reduced maximum specific growth rate of the EGDe rpsUG50C mutant is likely unrelated to SigB activation and potentially linked to impaired ribosomal function.

Whole genome analysis and cold adaptation strategies of Pseudomonas sivasensis W-6 isolated from the Napahai plateau wetland

Microbial communities of wetlands play key roles in the earth’s ecology and stability. To elucidate the cold adaptation mechanisms of bacteria in plateau wetlands, we conducted comparative genomic analyses of Pseudomonas sivasensis and closely related lineages. The genome of P. sivasensis W-6, a cold-adapted bacterium isolated from the Napahai plateau wetland, was sequenced and analyzed. The genome length was 6,109,123 bp with a G+C content of 59.5%. Gene prediction yielded 5360 protein-coding sequences, 70 tRNAs, 24 gene islands, and 2 CRISPR sequences. The isolate contained evidence of horizontal gene transfer events during its evolution. Two prophages were predicted and indicated that W-6 was a lysogen. The cold adaptation of the W-6 strain showed psychrophilic rather than psychrotrophic characteristics. Cold-adapted bacterium W-6 can utilize glycogen and trehalose as resources, associated with carbohydrate-active enzymes, and survive in a low-temperature environment. In addition, the cold-adapted mechanisms of the W-6 included membrane fluidity by changing the unsaturated fatty acid profile, the two-component regulatory systems, anti-sense transcription, the role played by rpsU genes in the translation process, etc. The genome-wide analysis of W-6 provided a deeper understanding of cold-adapted strategies of bacteria in environments. We elucidated the adaptive mechanism of the psychrophilic W-6 strain for survival in a cold environment, which provided a basis for further study on host-phage coevolution.

Amino acid substitutions in ribosomal protein RpsU enable switching between high fitness and multiple-stress resistance in Listeria monocytogenes

Microbial population heterogeneity contributes to differences in stress response between individual cells in a population, and can lead to the selection of genetically stable variants with increased stress resistance. We previously provided evidence that the multiple-stress resistant Listeria monocytogenes LO28 variant 15, carries a point mutation in the rpsU gene, resulting in an arginine-proline substitution in ribosomal protein RpsU (RpsU17Arg-Pro). Here, we investigated the trade-off between general stress sigma factor SigB-mediated stress resistance and fitness in variant 15 using experimental evolution. By selecting for higher fitness in two parallel evolving cultures, we identified two evolved variants: 15EV1 and 15EV2. Whole genome sequencing and SNP analysis showed that both parallel lines mutated in the same codon in rpsU as the original mutation resulting in RpsU17Pro-His (15EV1) and RpsU17Pro-Thr (15EV2). Using a combined phenotyping and proteomics approach, we assessed the resistance of the evolved variants to both heat and acid stress, and found that in both lines reversion to WT-like fitness also resulted in WT-like stress sensitivity. Proteome analysis of L. monocytogenes LO28 WT, variant 15, 15EV1, and 15EV2 revealed high level expression of SigB regulon members only in variant 15, whereas protein profiles of both evolved variants were highly similar to that of the LO28 WT. Experiments with constructed RpsU17Arg-Pro mutants in L. monocytogenes LO28 and EGDe, and RpsU17Arg-His and RpsU17Arg-Thr in LO28, confirmed that single amino acid substitutions in RpsU enable switching between multiple-stress resistant and high fitness states in L. monocytogenes.

Reduced vancomycin susceptibility and increased macrophage survival in Staphylococcus aureus strains sequentially isolated from a bacteraemic patient during a short course of antibiotic therapy.

The purpose of the present study was to determine the relatedness of Staphylococcus aureus strains successively isolated over a 7-day period from a single bacteraemic patient undergoing antibiotic treatment with vancomycin. The S. aureus strains had been isolated and sequenced previously. Antibiotic susceptibility testing, population analysis profiling, and lysostaphin sensitivity and phagocytic killing assays were used to characterize these clonal isolates. The seven isolates (MEH1-MEH7) were determined to belong to a common multilocus sequence type (MLST) and spa type. Within the third and fifth day of vancomycin treatment, mutations were observed in the vraS and rpsU genes, respectively. Population analysis profiles revealed that the initial isolate (MEH1) was vancomycin-susceptible S. aureus (VSSA), while those isolated on day 7 were mostly heteroresistant vancomycin-intermediate S. aureus (hVISA). Supporting these findings, MEH7 was also observed to be slower in growth, to have an increase in cell wall width and to have reduced sensitivity to lysostaphin, all characteristics of VISA and hVISA strains. In addition, MEH7, although phagocytosed at numbers comparable to the initial isolate, MEH1, survived in higher numbers in RAW 264.7 macrophages. Macrophages infected with MEH7 also released more TNF-α and IFN-1β. We report an increasing resistance to vancomycin coupled with daptomycin that occurred within approximately 3 days of receiving vancomycin and steadily increased until the infection was cleared with an alternative antibiotic therapy. This study reiterates the need for rapid, efficient and accurate detection of hVISA and VISA infections, especially in high-bacterial load, metastatic infections like bacteraemia.

Gene profiling-based phenotyping for identification of cellular parameters that contribute to fitness, stress-tolerance and virulence of Listeria monocytogenes variants

Microbial population heterogeneity allows for a differential microbial response to environmental stresses and can lead to the selection of stress resistant variants. In this study, we have used two different stress resistant variants of Listeria monocytogenes LO28 with mutations in the rpsU gene encoding ribosomal protein S21, to elucidate features that can contribute to fitness, stress-tolerance and host interaction using a comparative gene profiling and phenotyping approach. Transcriptome analysis showed that 116 genes were upregulated and 114 genes were downregulated in both rpsU variants. Upregulated genes included a major contribution of SigB-controlled genes such as intracellular acid resistance-associated glutamate decarboxylase (GAD) (gad3), genes involved in compatible solute uptake (opuC), glycerol metabolism (glpF, glpK, glpD), and virulence (inlA, inlB). Downregulated genes in the two variants involved mainly genes involved in flagella synthesis and motility. Phenotyping results of the two rpsU variants matched the gene profiling data including enhanced freezing resistance conceivably linked to compatible solute accumulation, higher glycerol utilisation rates, and better adhesion to Caco 2 cells presumably linked to higher expression of internalins. Also, bright field and electron microscopy analysis confirmed reduced flagellation of the variants. The activation of SigB-mediated stress defence offers an explanation for the multiple-stress resistant phenotype in rpsU variants.

Correlation of rpsU Gene Sequence Clusters and Biochemical Properties, Gc-Ms Spectra and Resistance Profiles of Clinical Burkholderia Spp. Isolates.

This study assessed the variation of phenotypic features of clinical isolates of Burkholderia spp. from common rpsU gene sequence clusters.A total of 41 clinical Burkholderia spp. isolates from German mucoviscidosis patients was subjected to rpsU gene sequencing. Biochemical assessment included the API systems 20 NE and 50 CHE as well as the Micronaut NF system. Fatty acid patterns were assessed using gas chromatography–mass spectrometry (GC–MS). Broth microdilution was used to identify minimum inhibitory concentrations.Five rpsU gene sequence clusters comprised more than one clinical isolate. Altogether, assignments to three species and seven clusters comprising more than one Burkholderia species were performed. Inhomogeneity of biochemical reactions within the clusters ranged from 0/28 to 45/50 reactions. The standard deviation for fatty acid distributions ranged from 0% to 11.5%. Minimum inhibitory concentrations within the clusters showed a wide variation but only minor differences between the clusters.Broad variations within identified rpsU gene sequence clusters regarding biochemical reactions, fatty acid patterns, and resistance patterns of clinical Burkholderia spp. isolates make the application of rpsU gene sequence analysis as a stand-alone procedure for discriminations within the Burkholderia cepacia complex unreliable.

Discrimination ofBurkholderia mallei/pseudomalleifromBurkholderia thailandensisby sequence comparison of a fragment of the ribosomal protein S21 (rpsU) gene

Discrimination of Burkholderia (B.) pseudomallei and B. mallei from environmental B. thailandensis is challenging. We describe a discrimination method based on sequence comparison of the ribosomal protein S21 (rpsU) gene.The rpsU gene was sequenced in ten B. pseudomallei, six B. mallei, one B. thailandensis reference strains, six isolates of B. pseudomallei, and 37 of B. thailandensis. Further rpsU sequences of six B. pseudomallei, three B. mallei, and one B. thailandensis were identified via NCBI GenBank. Three to four variable base-positions were identified within a 120-base-pair fragment, allowing discrimination of the B. pseudomallei/mallei-cluster from B. thailandensis, whose sequences clustered identically. All B. mallei and three B. pseudomallei sequences were identical, while 17/22 B. pseudomallei strains differed in one nucleotide (78A>C). Sequences of the rpsU fragment of 'out-stander' reference strains of B. cepacia, B. gladioli, B. plantarii, and B. vietnamensis clustered differently.Sequence comparison of the described rpsU gene fragment can be used as a supplementary diagnostic procedure for the discrimination of B. mallei/pseudomallei from B. thailandensis as well as from other species of the genus Burkholderia, keeping in mind that it does not allow for a differentiation between B. mallei and B. pseudomallei.

Rapid, Specific Detection of Enterobacter sakazakii in Infant Formula Using a Real-Time PCR Assay

Enterobacter sakazakii is a rare cause of invasive infection with high mortality rates in neonates. Powdered milk–based infant formulas have been associated with the E. sakazakii–related outbreaks in premature or other immunocompromised infants. In this study, an assay was developed for the specific detection of E. sakazakii in infant formula using an application of the fluorogenic 5′ nuclease assay (TaqMan). A set of primers and probe was designed using the E. sakazakii partial macromolecular synthesis operon: the rpsU gene 3′ end and the primase (dnaG) gene 5′ end. The specificity of the assay was evaluated using 68 Enterobacter and 55 non-Enterobacter strains. The newly developed assay enables us to detect 100 CFU/ml in pure culture and in reconstituted infant formula in 50 cycles of PCR without enrichment. The assay was specific enough to discriminate E. sakazakii from all other Enterobacter and non-Enterobacter strains tested. The developed real-time PCR assay could save up to 5 days and eliminate the need for plating samples on selective or diagnostic agars and for biochemical confirmation steps. The real-time PCR assay could be used to rapidly screen infant formula samples for E. sakazakii and would be a boon to food industries and regulatory agencies.

Temperature-dependent regulation of the ribosomal small-subunit protein S21 in the cyanobacterium Anabaena variabilis M3.

The rpsU gene, which encodes the ribosomal small-subunit protein S21 in Anabaena, is not a part of the macromolecular-synthesis operon as in most enterobacteria but rather is located downstream of the rbpA1 gene, which encodes an RNA-binding protein. Two types of transcripts were detected for this gene cluster. The level of the major rbpA1-rpsU transcript was about 10 times higher at 22 degrees C than at 38 degrees C, whereas the minor monocistronic rpsU transcript was more abundant at the higher temperature. The level of the S21 protein in relation to total protein was three times lower at 38 degrees C than at 22 degrees C. Analysis of isolated ribosomes indicated that S21 was present at an equimolar ratio with regard to other ribosomal proteins at 22 degrees C but that its level decreased with temperature. Conversely, the relative abundance of S5 increased with temperature. A decrease in the level of S21 at high temperature was also found in Synechocystis, in which rpsU is located downstream of the rrn operon. These results suggest that S21 is involved in the adaptation to changes in temperature in cyanobacteria.

Disruption Analysis of the Gene for a Cold-Regulated RNA-Binding Protein, rbpA1, in Anabaena: Cold-Induced Initiation of the Heterocyst Differentiation Pathway

A cold-regulated operon, rbpA1-rpsU, encodes an RNA-binding protein and a ribosomal protein in Anabaena variabilis M3. The level of expression of this gene cluster was about ten times higher at temperatures below 30 degrees C than at 38 degrees C. To study the role of the RbpA1 protein in vivo, we constructed insertional disruptants of rbpA1. These strains were totally devoid of RbpA1 protein but contained a normal level of the ribosomal protein S21, a product of the rpsU gene. The disruptants were morphologically normal at 38 degrees C, but at 22 degrees C they produced unusual cells at a low frequency. These cells were probably at an initial stage of proheterocyst formation. Various molecular events that are related to heterocyst initiation, namely, excision of the 11-kbp DNA element in nifD and the accumulation of transcripts of xisA and hetR, also occurred in the disruptants at 22 degrees C in the presence of nitrate ions, but these events did not occur in the presence of ammonium ions or at 38 degrees C. The results suggest that RbpA1 is required for enhanced repression of heterocyst initiation at low temperatures in the presence of nitrate. Possible mechanisms of the action of RabA1 are discussed.

An Escherichia coli cis-acting antiterminator sequence: the dnaG nut site.

The Escherichia coli rpsU-dnaG-rpoD operon contains an internal transcription terminator T1 located in the intergenic region between the rpsU and dnaG genes (Smiley et al. 1982). By cloning T1 as a small 127 bp fragment into the terminator probe plasmid pDR720 between the trp operator promoter and the assayable galK gene, it was shown that T1 acts as a strong transcription terminator, comparable in strength to the 3' operon terminator T2. However, an operon sequence that occurs 5' to T1 within the coding region of the rpsU gene and which has homology with the lambda nut site, (Lupski et al. 1983) when placed 5' to T1 in the pDR720 plasmid construct, modifies transcription through T1 allowing expression of the galK gene. This sequence, called the dnaG nut site also modifies the termination activity of the external operon terminator T2. It is proposed that the dnaG nut site is a cis-acting element of an antitermination system in E. coli.

Possible new genes as revealed by molecular analysis of a 5-kb Escherichia coli chromosomal region 5′ to the rpsU-dnaG-rpoD macromolecular-synthesis operon

The complete nucleotide sequence of 5-kb DNA fragment immediately 5′ to the rpsU-dnaG-rpoD macro-molecular-synthesis operon in Escherichia coli has been determined. It encodes for six open reading frames. Transcriptional and translational analysis have shown that three of them ( orf x, orf z1 and orf z2) are expressed in exponentially growing E. coli cells. The orf x, directly 5′ to the rpsU gene but transcribed in the opposite direction, may be part of the rpsU-dnaG-rpoD macromolecular-synthesis operon.