Comparative genomics of the proteostasis network in extreme acidophiles.

Extreme acidophiles thrive in harsh environments characterized by acidic pH, high concentrations of dissolved metals and high osmolarity. Most of these microorganisms are chemolithoautotrophs that obtain energy from low redox potential sources, such as the oxidation of ferrous ions. Under these conditions, the mechanisms that maintain homeostasis of proteins (proteostasis), as the main organic components of the cells, are of utmost importance. Thus, the analysis of protein chaperones is critical for understanding how these organisms deal with proteostasis under such environmental conditions. In this work, using a bioinformatics approach, we performed a comparative genomic analysis of the genes encoding classical, periplasmic and stress chaperones, and the protease systems. The analysis included 35 genomes from iron- or sulfur-oxidizing autotrophic, heterotrophic, and mixotrophic acidophilic bacteria. The results showed that classical ATP-dependent chaperones, mostly folding chaperones, are widely distributed, although they are sub-represented in some groups. Acidophilic bacteria showed redundancy of genes coding for the ATP-independent holdase chaperones RidA and Hsp20. In addition, a systematically high redundancy of genes encoding periplasmic chaperones like HtrA and YidC was also detected. In the same way, the proteolytic ATPase complexes ClpPX and Lon presented redundancy and broad distribution. The presence of genes that encoded protein variants was noticeable. In addition, genes for chaperones and protease systems were clustered within the genomes, suggesting common regulation of these activities. Finally, some genes were differentially distributed between bacteria as a function of the autotrophic or heterotrophic character of their metabolism. These results suggest that acidophiles possess an abundant and flexible proteostasis network that protects proteins in organisms living in energy-limiting and extreme environmental conditions. Therefore, our results provide a means for understanding the diversity and significance of proteostasis mechanisms in extreme acidophilic bacteria.

Long-term (18 Years) Results of Patients With Long-segment Barrett Esophagus Submitted to Acid Suppression-duodenal Diversion Operation: Better Than Nissen Fundoplication?

OBJECTIVE: To determine late results of AS-DD procedure in long-segment (LSBE) and extralong-segment BE (ELSBE) using subjective and objective measurements to ascertain the histological impact over intestinal metaplasia (IM) and progression to EAC. SUMMARY OF BACKGROUND DATA: Barrett esophagus (BE) is a known precursor of esophageal adenocarcinoma (EAC), and Nissen fundoplication has proven to be unable to stop mixed reflux among them. Our group proposed a surgical procedure that handles pathophysiological changes responsible for BE. METHODS: This prospective study included 127 LSBE and ELSBE subjects submitted to clinical and functional analyses. They were presented to selective vagotomy, fundoplication, partial gastrectomy with Roux-en-Y reconstruction. The changes in IM were determined in both groups. RESULTS: Follow-up was completed at a mean of 18 years in 81% of the cases. Visick I-II scores were seen in 88% of LSBE and 65% in ELSBE ( P < 0.01). There was significant healing of erosive esophagitis and esophageal peptic ulcers, and strictures were resolved in 71%. There was 38% of IM regression in LSBE. Two cases in each group progressed to EAC at a mean of 15 years. Pathologic acid reflux was abolished in 91% and duodenal in 100%. There was a regression of low-grade dysplasia to IM in 80%. CONCLUSIONS: AS-DD permanently eliminates pathologic refluxate to the esophagus. The progression to HGD/EAC is lower compared to medical treatment, with an 8-fold reduction in LSBE and 2.2-fold in ELSBE. AS-DD seems to influence IM behaviors, and it is a tool that could reduce and delay progression to EAC.

Oxidative stress strongly restricts the effect of codon choice on the efficiency of protein synthesis in Escherichia coli.

Introduction: The response of enterobacteria to oxidative stress is usually considered to be regulated by transcription factors such as OxyR and SoxR. Nevertheless, several reports have shown that under oxidative stress the levels, modification and aminoacylation of tRNAs may be altered suggesting a role of codon bias in regulation of gene expression under this condition. Methods: In order to characterize the effects of oxidative stress on translation elongation we constructed a library of 61 plasmids, each coding for the green fluorescent protein (GFP) translationally fused to a different set of four identical codons. Results: Using these reporters, we observed that GFP production levels vary widely (~15 fold) when Escherichia coli K-12 is cultured in minimal media as a consequence of codon choice variations. When bacteria are cultured under oxidative stress caused by paraquat the levels of GFP produced by most clones is reduced and, in contrast to control conditions, the range of GFP levels is restricted to a ~2 fold range. Restricting elongation of particular sequences does not increase the range of GFP production under oxidative stress, but altering translation initiation rates leads to an increase in this range. Discussion: Altogether, our results suggest that under normal conditions the speed of translation elongation is in the range of the speed of initiation and, consequently, codon choice impacts the speed of protein synthesis. In contrast, under oxidative stress translation initiation becomes much slower than elongation, limiting the speed of translation such that codon choice has at most only subtle effects on the overall output of translation.

Long-term (17 years) subjective and objective evaluation of the durability of laparoscopic Heller esophagomyotomy in patients with achalasia of the esophagus (90% of follow-up): a real challenge to POEM.

INTRODUCTION: Laparoscopic Heller Myotomy (LHM) with partial anterior or posterior fundoplication is the standard surgical procedure for treating achalasia patients. The results reported are mainly based on symptomatic evaluations and have less than 5 years of follow-up and none more than ten. OBJECTIVE: To determine the late results of LHM, performing endoscopic, histologic, manometric, and functional studies in addition to clinical evaluations. MATERIALS AND METHODS: Eighty-nine consecutive patients were included in a prospective study from 1993 to 2008. Inclusion criteria corresponded to achalasia patients with Types I to III (radiological evaluation). Exclusion criteria included patients with grade IV, patients with previous procedures (surgical or endoscopic), or giant hiatal hernia. They were submitted to a radiological evaluation, over two endoscopic procedures with biopsy samples, manometric assessments, and 24-h pH monitoring late after surgery. RESULTS: There was no operative mortality nor postoperative complications. The average hospital stay was two days. Nine patients (10.1%) were lost from follow-up. The mean late follow-up was 17 years (10-26). Visick I and II (success) corresponded to 78.7% of patients and grades III-IV (failure) to 21.3%, mainly due to gastroesophageal reflux disease (GERD). Manometric evaluations showed a significant and permanent decrease in lower esophageal sphincter pressure (LESP). 24-h pH monitoring was normal among Visick I patients and showed pathologic acid reflux in patients with GERD. Two patients (2.5%) developed Barrett's esophagus. Squamous-cell carcinoma (SCC) appeared in three patients (3.7%). CONCLUSION: LHM controlled symptoms in 79% of achalasia patients very late (17 years) after surgery. This was corroborated by endoscopic, manometric, and functional studies. GERD symptoms developed in 18.7% and SCC in 3.7% in previously asymptomatic patients. Endoscopic surveillance at regular intervals is recommended for all patients who have had surgery. These very long-term results are a real challenge to POEM endoscopic treatment. Unique Identifying Registration Number 3743.

Synonymous mutations in the phosphoglycerate kinase 1 gene induce an altered response to protein misfolding in Schizosaccharomyces pombe.

Background: Proteostasis refers to the processes that regulate the biogenesis, folding, trafficking, and degradation of proteins. Any alteration in these processes can lead to cell malfunction. Protein synthesis, a key proteostatic process, is highly-regulated at multiple levels to ensure adequate adaptation to environmental and physiological challenges such as different stressors, proteotoxic conditions and aging, among other factors. Because alterations in protein translation can lead to protein misfolding, examining how protein translation is regulated may also help to elucidate in part how proteostasis is controlled. Codon usage bias has been implicated in the fine-tuning of translation rate, as more-frequent codons might be read faster than their less-frequent counterparts. Thus, alterations in codon usage due to synonymous mutations may alter translation kinetics and thereby affect the folding of the nascent polypeptide, without altering its primary structure. To date, it has been difficult to predict the effect of synonymous mutations on protein folding and cellular fitness due to a scarcity of relevant data. Thus, the purpose of this work was to assess the effect of synonymous mutations in discrete regions of the gene that encodes the highly-expressed enzyme 3-phosphoglycerate kinase 1 (pgk1) in the fission yeast Schizosaccharomyces pombe. Results: By means of systematic replacement of synonymous codons along pgk1, we found slightly-altered protein folding and activity in a region-specific manner. However, alterations in protein aggregation, heat stress as well as changes in proteasome activity occurred independently of the mutated region. Concomitantly, reduced mRNA levels of the chaperones Hsp9 and Hsp16 were observed. Conclusion: Taken together, these data suggest that codon usage bias of the gene encoding this highly-expressed protein is an important regulator of protein function and proteostasis.

Sarcomatoid carcinoma of the pancreas - a rare tumor with an uncommon presentation and course: A case report and review of literature.

BACKGROUND: Sarcomatoid carcinoma of the pancreas (SCP) is a rare type of pancreatic neoplasm, and only a few cases have been described in the literature. Histologically, it is composed mostly of atypical spindle cells with apparent sarcomatous features. CASE SUMMARY: This is a report of a 61-year-old Chilean woman who underwent medical investigation for acute abdominal pain. Computed tomography identified a solid tumor in the tail of the pancreas with features suspicious of malignancy. En-bloc distal pancreatectomy and splenectomy were performed to excise the tumor. Histopathology and immunohistochemistry were confirmatory of sarcomatoid carcinoma with lymphovascular invasion. After surgery, the patient did not receive chemotherapy. Previous studies indicate a poor prognosis for this type of malignancy. However, our patient has survived for 35 mo with no recurrence to date. CONCLUSION: The case presented herein is a patient with an SCP with a rare presentation and long-term survival after surgery despite not receiving adjuvant chemotherapy.

Redox stress response and UV tolerance in the acidophilic iron-oxidizing bacteria Leptospirillum ferriphilum and Acidithiobacillus ferrooxidans.

The oxidative stress response represents a sum of antioxidative mechanisms that are essential for determining the adaptation and abundance of microorganisms in the environment. Leptospirillum ferriphilum and Acidithiobacillus ferrooxidans are chemolithotrophic bacteria that obtain their energy from the oxidation of ferrous ion. Both microorganisms are important for bioleaching of sulfidic ores and both are tolerant to high levels of heavy metals and other factors that can induce oxidative stress. In this work, we compared the tolerance and response of L. ferriphilum and At. ferrooxidans to Fe3+, H2O2, K2CrO4, and UV-C radiation. We evaluated growth, generation of reactive oxygen species (ROS), oxidative damage to lipid membranes and DNA, and the activity of antioxidative proteins in cells exposed to these stressors. L. ferriphilum had higher cell density, lower ROS content and less lipid and DNA damage than At. ferrooxidans. Consistent with this, the activity levels of thioredoxin and superoxide dismutase in L. ferriphilum were upregulated and higher than in At. ferrooxidans. This indicated that L. ferriphilum has a higher capacity to respond to oxidative stress and to manage redox homeostasis. This capacity could largely contribute to the high abundance of this species in natural and anthropogenic sites.

LESSONS LEARNED ANALYZING COMPLICATIONS AFTER LAPAROSCOPIC TOTAL GASTRECTOMY FOR GASTRIC CANCER.

BACKGROUND: Laparoscopic surgery has been gradually accepted as an option for the surgical treatment ofgastric cancer. There are still points that are controversial or situations that are eventually associated with intra-operative difficulties or postoperative complications. AIM: To establish the relationship between the difficulties during the execution of total gastrectomy and the occurrence of eventual postoperative complications. METHOD: The operative protocols and postoperative evolution of 74 patients operated for gastriccancer, who were subjected to laparoscopic total gastrectomy (inclusion criteria) were reviewed. The intraoperative difficulties recorded in the operative protocol and postoperative complications of a surgical nature wereanalyzed (inclusion criteria). Postoperative medical complications were excluded (exclusion criteria). For the discussion, an extensive bibliographical review was carried out. RESULTS: Intra-operative difficulties or complications reported correspond to 33/74 and of these; 18 events (54.5%) were related to postoperative complications and six were absolutely unexpected. The more frequent were leaks of the anastomosis and leaks of the duodenal stump; however, other rare complications were observed. Seven were managed with conservative measures and 17 (22.9%) required surgical re-exploration, with a postoperative mortality of two patients (2.7%). CONCLUSION: We have learned that there are infrequent and unexpected complications; the treating team must be mindful of and, in front of suspicion of complications, anappropriate decision must be done which includes early re-exploration. Finally, after the experience reported, some complications should be avoided.

Deciphering the Role of Multiple Thioredoxin Fold Proteins of Leptospirillum sp. in Oxidative Stress Tolerance.

Thioredoxin fold proteins (TFPs) form a family of diverse proteins involved in thiol/disulfide exchange in cells from all domains of life. Leptospirillum spp. are bioleaching bacteria naturally exposed to extreme conditions like acidic pH and high concentrations of metals that can contribute to the generation of reactive oxygen species (ROS) and consequently the induction of thiol oxidative damage. Bioinformatic studies have predicted 13 genes that encode for TFP proteins in Leptospirillum spp. We analyzed the participation of individual tfp genes from Leptospirillum sp. CF-1 in the response to oxidative conditions. Genomic context analysis predicted the involvement of these genes in the general thiol-reducing system, cofactor biosynthesis, carbon fixation, cytochrome c biogenesis, signal transduction, and pilus and fimbria assembly. All tfp genes identified were transcriptionally active, although they responded differentially to ferric sulfate and diamide stress. Some of these genes confer oxidative protection to a thioredoxin-deficient Escherichia coli strain by restoring the wild-type phenotype under oxidative stress conditions. These findings contribute to our understanding of the diversity and complexity of thiol/disulfide systems, and of adaptations that emerge in acidophilic microorganisms that allow them to thrive in highly oxidative environments. These findings also give new insights into the physiology of these microorganisms during industrial bioleaching operations.

Modification of Transfer RNA Levels Affects Cyclin Aggregation and the Correct Duplication of Yeast Cells.

Codon usage bias (the preferential use of certain synonymous codons (optimal) over others is found at the organism level (intergenomic) within specific genomes (intragenomic) and even in certain genes. Whether it is the result of genetic drift due to GC/AT content and/or natural selection is a topic of intense debate. Preferential codons are mostly found in genes encoding highly-expressed proteins, while lowly-expressed proteins usually contain a high proportion of rare (lowly-represented) codons. While optimal codons are decoded by highly expressed tRNAs, rare codons are usually decoded by lowly-represented tRNAs. Whether rare codons play a role in controlling the expression of lowly- or temporarily-expressed proteins is an open question. In this work we approached this question using two strategies, either by replacing rare glycine codons with optimal counterparts in the gene that encodes the cell cycle protein Cdc13, or by overexpression the tRNA Gly that decodes rare codons from the fission yeast, Schizosaccharomyces pombe. While the replacement of synonymous codons severely affected cell growth, increasing tRNA levels affected the aggregation status of Cdc13 and cell division. These lead us to think that rare codons in lowly-expressed cyclin proteins are crucial for cell division, and that the overexpression of tRNA that decodes rare codons affects the expression of proteins containing these rare codons. These codons may be the result of the natural selection of codons in genes that encode lowly-expressed proteins.

Lessons learned analyzing complications after laparoscopic total gastrectomy for gastric cancer / Lições aprendidas analisando complicações da gastrectomia total laparoscópica para câncer gástrico

ABSTRACT Background: Laparoscopic surgery has been gradually accepted as an option for the surgical treatment ofgastric cancer. There are still points that are controversial or situations that are eventually associated with intra-operative difficulties or postoperative complications. Aim: To establish the relationship between the difficulties during the execution of total gastrectomy and the occurrence of eventual postoperative complications. Method: The operative protocols and postoperative evolution of 74 patients operated for gastriccancer, who were subjected to laparoscopic total gastrectomy (inclusion criteria) were reviewed. The intraoperative difficulties recorded in the operative protocol and postoperative complications of a surgical nature wereanalyzed (inclusion criteria). Postoperative medical complications were excluded (exclusion criteria). For the discussion, an extensive bibliographical review was carried out. Results: Intra-operative difficulties or complications reported correspond to 33/74 and of these; 18 events (54.5%) were related to postoperative complications and six were absolutely unexpected. The more frequent were leaks of the anastomosis and leaks of the duodenal stump; however, other rare complications were observed. Seven were managed with conservative measures and 17 (22.9%) required surgical re-exploration, with a postoperative mortality of two patients (2.7%). Conclusion: We have learned that there are infrequent and unexpected complications; the treating team must be mindful of and, in front of suspicion of complications, anappropriate decision must be done which includes early re-exploration. Finally, after the experience reported, some complications should be avoided.

RESUMO Racional: A cirurgia laparoscópica tem sido gradualmente aceita como opção para o tratamento cirúrgico do câncer gástrico. Ainda existem pontos controversos ou situações eventualmente associadas a dificuldades intra-operatórias ou complicações pós-operatórias. Objetivo: Estabelecer a relação entre as dificuldades durante a execução da gastrectomia total e a ocorrência de eventuais complicações pós-operatórias. Método: Foram revisados ​​os protocolos operatórios e a evolução pós-operatória de 74 pacientes operados por câncer gástrico, submetidos à gastrectomia total laparoscópica (critérios de inclusão). Foram analisadas as dificuldades intraoperatórias registradas no protocolo operatório e as complicações pós-operatórias de natureza cirúrgica (critérios de inclusão). As complicações médicas pós-operatórias foram excluídas (critérios de exclusão). Para a discussão, foi realizada extensa revisão bibliográfica. Resultados: Dificuldades ou complicações intraoperatórias relatadas corresponderam a 33/74 e destas 18 (54,5%) foram relacionadas com complicações pós-operatórias e seis absolutamente inesperadas. As mais frequentes foram vazamentos da anastomose e do coto duodenal; no entretanto, outras complicações raras foram observadas. Sete foram tratados com medidas conservadoras e 17 (22,9%) necessitaram de re-exploração cirúrgica, com mortalidade pós-operatória de dois pacientes (2,7%). Conclusão: Aprendemos que existem complicações infrequentes e inesperadas; a equipe de tratamento deve estar atenta e diante da suspeita de complicação, decisão apropriada pode incluir uma nova exploração precoce. Finalmente, após a experiência relatada, algumas complicações devem ser evitadas.

Clinical, Endoscopic, and Histologic Findings at the Distal Esophagus and Stomach Before and Late (10.5 Years) After Laparoscopic Sleeve Gastrectomy: Results of a Prospective Study with 93% Follow-Up.

OBJECTIVE: Perform a prospective study based on sequential clinical, endoscopic, and histologic evaluations of the foregut late after laparoscopic sleeve gastrectomy (LSG) in obese patients. After LSG, several studies have suggested an increase in the incidence of clinical gastroesophageal reflux (GERD) while others have reported an improvement but based mainly on clinical questionnaires. METHODS: Prospective study of 104 consecutive patients submitted to LSG. Several postoperative endoscopic and histologic evaluations of the esophagogastric junction (EGJ) and the gastric tube (GT) were performed and correlated with symptomatic findings. RESULTS: According to clinical preoperative findings, patients were divided into non-refluxers (Group I) and refluxers (Group II). Seven patients were unreachable, leaving 97 (93%) for late evaluation. Among Group I, 58.5% developed de novo GERD, while in Group II just 13.6% showed the disappearance of them. Endoscopic evaluations showed progressive deterioration of the EGJ in Group I, with the development of erosive esophagitis (EE), hiatal hernia (HH), and dilated cardia in a large proportion of them. In the GT, the presence of bile was seen in 40%, and an open immobile pylorus was detected in 82%. Short-segment Barrett's esophagus (BE) appeared in 4%. CONCLUSIONS: Patients submitted to LSG showed a significant and progressive increase in the presence of "de novo" GERD. Also, an increased duodenogastric reflux was seen through an open and immobile pylorus. Therefore, based on these results, it seems like LSG is a "pro-reflux" surgical procedure, which should be continuously evaluated late after surgery.

Patients Remain at High Risk of Gallstones Development Late (10 y) After Sleeve Gastrectomy?

BACKGROUND: Sleeve gastrectomy (SG) is an established bariatric procedure which produces substantial and rapid weight loss and hence can lead to an increase in gallstones development. OBJECTIVES: To demonstrate the early and late appearance of gallstones after SG. MATERIALS AND METHODS: A prospective protocol was established in consecutive patients submitted to SG. Clinical and ultrasound evaluations were performed early (1 to 2 y) and late after surgery (over 6 y). RESULTS: From 109 patients included, 13 (13.1%) had a previous and 10 (10.1%) had simultaneous cholecystectomy at the time of SG. Therefore, 86 patients were submitted to surveillance. Seven patients were unreachable, leaving 79 patients for late follow-up. Forty-five patients (57%) had alithiasic gallbladder late after surgery, whereas 34 patients (43%) showed appearance of gallstone. From them, 53% developed gallstones late after surgery (mean, 7.5 y). Among the group with early development of stones, 69% were symptomatic and in the latter group only 17%. CONCLUSIONS: Study with 92% of follow-up late after SG demonstrated a 43% development of gallstones: half earlier and half late after surgery. We emphasize the need for late control to detect the real appearance of gallstones after SG.

Long-term (15-year) objective evaluation of 150 patients after laparoscopic Nissen fundoplication.

INTRODUCTION: Laparoscopic Nissen fundoplication is the preferred operative treatment for patients with gastroesophageal reflux disease. The most recent published results only refer to clinical evaluations and few discuss objective measurements. Our purpose was to determine the late results of laparoscopic Nissen fundoplication, performing clinical, endoscopic, histologic, and functional studies. MATERIAL AND METHODS: A total of 179 patients were included in a prospective study. All had gastroesophageal reflux disease symptoms of at least 5-year duration, daily dependence on proton pump inhibitors, and a type I hiatal hernia less than 5 cm. Exclusion criteria included Barrett's esophagus, hiatal hernia >5 cm, failed antireflux surgery, and obesity (body mass index >30). We performed a radiologic study, 3 or more endoscopic procedures with biopsy samples of the antrum and esophagogastric junction, esophageal manometry, and 24-hour pH monitoring. RESULTS: We found that 4 patients (2.2%) died 3-4 years after operation from nonoperatiove reasons. A total of 25 patients (14%) were lost to follow-up, and 150 patients (83.8%) submitted to late objective evaluations (15 years). Visick I-II symptoms were observed in 79.3% and III-IV (failures) in 20.7%. Endoscopy showed a normal positioning of the esophagogastric junction in the Visick I-II patients and a type III cardia or hiatal hernia with erosive esophagitis in Visick III-IV patients. Short-segment Barrett's esophagus developed in 5.3% of patients. Lower esophageal sphincter pressure remained increased over the preoperative value in all groups. The 24-hour pH monitoring also was decreased over the preoperative value in Visick I-II patients but showed no significant change in Visick III-IV patients. Carditis at the esophagogastric junction regressed to fundic mucosa in 50% of Visick I-II patients. CONCLUSION: Laparoscopic Nissen fundoplication produces control of symptoms in 80% of patients late (up to 15 years) after surgeries corroborated by endoscopic, histologic examinations, and functional studies. It is essential to perform these objective evaluations to demonstrate the "antireflux effect" after laparoscopic Nissen fundoplication.

The Type IV Secretion System of ICEAfe1: Formation of a Conjugative Pilus in Acidithiobacillus ferrooxidans.

The dispersal of mobile genetic elements and their gene cargo relies on type IV secretion systems (T4SS). In this work the ICEAfe1 Tra-type T4SS nanomachine, encoded in the publicly available genome of Acidithiobacillus ferrooxidans ATCC 23270TY, was characterized in terms of its organization, conservation, expression and mating bridge formation. Twenty-one conjugative genes grouped in four genetic clusters encode the ICEAfe1 T4SS, containing all the indispensable functions for the formation and stabilization of the pili and for DNA processing. The clusters' organization resembles that of other mobile genetic elements (such as plasmids and integrative and conjugative elements-ICEs). Sequence conservation, genetic organization and distribution of the tra system in the genomes of other sequenced Acidithiobacillus spp. suggests that the ICEAfe1 T4SS could mediate the lateral gene transfer between related bacteria. All ICEAfe1 T4SS genes are transcriptionally active and expressed from four independent operons. The transcriptional levels of selected marker genes increase in response to Mitomycin C treatment, a DNA damage elicitor that has acknowledged stimulatory effects on excision rates and gene expression of other ICEs, including ICEAfe1. Using a tailor-made pilin-antiserum against ICEAfe1 T4SS TraA pilin and epifluorescence microscopy, the presence of the conjugative pili on the cell surface of A. ferrooxidans could be demonstrated. Additionally, immunodetection assays, by immunogold, allowed the identification of pili-like extracellular structures. Together, the results obtained in this work demonstrate that the ICEAfe1 T4SS is phylogenetically conserved within the taxon, is expressed at mRNA and protein levels in vivo in the A. ferrooxidans type strain, and produces a pili-like structure of extracellular and intercellular localization in this model acidophile, supporting its functionality. Additional efforts will be required to prove conjugation of the ICEAfe1 or parts of this element through the cognate T4SS.

Codon usage and modular interactions between messenger RNA coding regions and small RNAs in Escherichia coli.

BACKGROUND: Small RNAs (sRNAs) are key regulators of gene expression in bacteria. In addition to modulating translation initiation, sRNAs can interact with mRNA coding regions to regulate mRNA stability and translation efficiency, enhancing or impeding progression of the ribosome along the mRNA. Since most amino acids are decoded by more than one codon (synonymous) we asked as to whether there is a codon bias in the interaction of sRNAs with coding regions of mRNAs. Therefore, we explored whether there are differences in codon usage or tRNA availability according to whether an mRNA is regulated by sRNAs or not. We also explored these parameters in the coding interaction regions in mRNAs. We focused our analysis on sRNAs that regulate multiple mRNAs. RESULTS: We found differences in codon adaptation index and tRNA adaptation index between sRNA-regulated and non-sRNA-regulated mRNAs. Interestingly, the sRNA-mRNA interacting regions tended to be enriched in unpreferred codons decoded by scarce tRNAs. We also found that sRNAs with multiple targets often contained modular segments capable of recognizing conserved motifs among these mRNAs. CONCLUSIONS: Our results show that sRNAs in E. coli tend to recognize mRNA coding regions in which the ribosome is predicted to advance at low speeds. Identified motifs in interacting regions are conserved among mRNAs that are recognized by the same sRNA.

Codon usage revisited: Lack of correlation between codon usage and the number of tRNA genes in enterobacteria.

It is widely believed that if a high number of genes are found for any tRNA in a rapidly replicating bacteria, then the cytoplasmic levels of that tRNA will be high and an open reading frame containing a higher frequency of the complementary codon will be translated faster. This idea is based on correlations between the number of tRNA genes, tRNA concentration and the frequency of codon usage observed in a limited number of strains as well as from the fact that artificially changing the number of tRNA genes alters translation efficiency and consequently the amount of properly folded protein synthesized. tRNA gene number may greatly vary in a genome due to duplications, deletions and lateral transfer which in turn would alter the levels and functionality of many proteins. Such changes are potentially deleterious for fitness and as a result it is expected that changes in tRNA gene numbers should be accompanied by a modification of the frequency of codon usage. In contrast to this model, when comparing the number of tRNA genes and the frequency of codon usage of several Salmonella enterica and Escherichia coli strains we found that changes in the number of tRNA genes are not correlated to changes in codon usage. Furthermore, these changes are not correlated with a change in the efficiency of codon translation. These results suggest that once a genome gains or loses tRNA genes, it responds by modulating the concentrations of tRNAs rather than modifying its frequency of codon usage.