A single-arm, prospective study comparing translaryngeal ultrasonography with direct visualisation by flexible laryngoscopy for vocal cord assessment in patients undergoing oesophagectomy or mediastinoscopy.

Background and Aims: Translaryngeal ultrasonography (TLUSG) for diagnosis of vocal cord palsy, a relatively new, safe and noninvasive bedside technique with minimal risk of respiratory infection transmission, has been effective in patients with thyroid disease. We studied its use as an alternative method to visual inspection by flexible laryngoscopy (FL) for vocal cord assessment in patients undergoing thoracic surgeries. Methods: After Institutional Ethics Committee approval and trial registration, in this single-arm, prospective study, the vocal cord function of 110 patients who underwent either total oesophagectomy or mediastinoscopy was assessed immediately after extubation by both FL and TLUSG. A follow-up assessment was done by laryngoscopy using Hopkin's endoscope (HL) and a repeat TLUSG. The primary outcome was the concordance between direct visualisation (FL or HL) and TLUSG. Results: Vocal cords were successfully visualised by TLUSG in 90% of male and all female patients. Findings of FL and TLUSG done at the first assessment matched in 89 (86.4%) out of 103 patients, and the degree of concordance was 0.69 (95% confidence interval [CI] =0.52-0.83). At the second assessment, HL and TLUSG findings matched in 83 (94.3%) out of 88 patients, and the degree of concordance was 0.89 (95% CI = 0.77-0.98). Conclusion: TLUSG is an effective noninvasive alternative to direct visualisation for vocal cord assessment in both male and female patients undergoing thoracic surgery.

A haem-sequestering plant peptide promotes iron uptake in symbiotic bacteria.

Symbiotic partnerships with rhizobial bacteria enable legumes to grow without nitrogen fertilizer because rhizobia convert atmospheric nitrogen gas into ammonia via nitrogenase. After Sinorhizobium meliloti penetrate the root nodules that they have elicited in Medicago truncatula, the plant produces a family of about 700 nodule cysteine-rich (NCR) peptides that guide the differentiation of endocytosed bacteria into nitrogen-fixing bacteroids. The sequences of the NCR peptides are related to the defensin class of antimicrobial peptides, but have been adapted to play symbiotic roles. Using a variety of spectroscopic, biophysical and biochemical techniques, we show here that the most extensively characterized NCR peptide, 24 amino acid NCR247, binds haem with nanomolar affinity. Bound haem molecules and their iron are initially made biologically inaccessible through the formation of hexamers (6 haem/6 NCR247) and then higher-order complexes. We present evidence that NCR247 is crucial for effective nitrogen-fixing symbiosis. We propose that by sequestering haem and its bound iron, NCR247 creates a physiological state of haem deprivation. This in turn induces an iron-starvation response in rhizobia that results in iron import, which itself is required for nitrogenase activity. Using the same methods as for L-NCR247, we show that the D-enantiomer of NCR247 can bind and sequester haem in an equivalent manner. The special abilities of NCR247 and its D-enantiomer to sequester haem suggest a broad range of potential applications related to human health.

A Mutant Era GTPase Suppresses Phenotypes Caused by Loss of Highly Conserved YbeY Protein in Escherichia coli.

Ribosome assembly is a complex fundamental cellular process that involves assembling multiple ribosomal proteins and several ribosomal RNA species in a highly coordinated yet flexible and resilient manner. The highly conserved YbeY protein is a single-strand specific endoribonuclease, important for ribosome assembly, 16S rRNA processing, and ribosome quality control. In Escherichia coli, ybeY deletion results in pleiotropic phenotypes including slow growth, temperature sensitivity, accumulation of precursors of 16S rRNA, and impaired formation of fully assembled 70S subunits. Era, an essential highly conserved GTPase protein, interacts with many ribosomal proteins, and its depletion results in ribosome assembly defects. YbeY has been shown to interact with Era together with ribosomal protein S11. In this study, we have analyzed a suppressor mutation, era(T99I), that can partially suppress a subset of the multiple phenotypes of ybeY deletion. The era(T99I) allele was able to improve 16S rRNA processing and ribosome assembly at 37°C. However, it failed to suppress the temperature sensitivity and did not improve 16S rRNA stability. The era(T99I) allele was also unable to improve the 16S rRNA processing defects caused by the loss of ribosome maturation factors. We also show that era(T99I) increases the GroEL levels in the 30S ribosome fractions independent of YbeY. We propose that the mechanism of suppression is that the changes in Era's structure caused by the era(T99I) mutation affect its GTP/GDP cycle in a way that increases the half-life of RNA binding to Era, thereby facilitating alternative processing of the 16S RNA precursor. Taken together, this study offers insights into the role of Era and YbeY in ribosome assembly and 16S rRNA processing events.

Prevalence, distribution and correlates of pain in patients with mucormycosis-A cross-sectional study.

Context: COVID-19 has led to a spate of rhino-orbital-cerebral mucormycosis cases in India, the epidemiology of which was least understood before. Only a few case series and case reports discuss the symptomatology of mucormycosis. Aims: The primary objective of our study was to estimate the prevalence of pain in patients with mucormycosis. The secondary objectives include the type, regional distribution, characteristics and determinants of pain in patients with mucormycosis. Settings and Design: A cross-sectional study was conducted on consecutive adult patients with mucormycosis in our hospital. Materials and Methods: Following recruitment, a preplanned written questionnaire that was tested for validity with peers, with closed-ended queries was filled on a sole visit by an anesthesia postgraduate based on the response by the patient. Statistical analysis used: Categorical variables were summarized as proportion and percentage. To compare quantitative variables, Chi-square test was used. Results: A total of 69 out of the 80 patients recruited complained of pain (P value = 0.468). A total of 76.8% of patients had pain in the supraorbital region with 84% of the patients complaining of throbbing pain and 98.6% needing analgesics. Gender, pre-existing diabetes mellitus, organs affected due to mucormycosis, prior steroid usage, prior COVID illness, surgical intervention, and previous experience of pain in the same region had no influence on the presence or severity of pain. Conclusions: Pain is one of the presenting symptoms in patients with mucormycosis which is usually in the supraorbital and maxillary region, nociceptive type and throbbing in nature, and moderate to severe in intensity usually managed with simple analgesics.

The Mutant ßE202K Sliding Clamp Protein Impairs DNA Polymerase III Replication Activity.

Expression of the Escherichia coli dnaN-encoded ß clamp at ≥10-fold higher than chromosomally expressed levels impedes growth by interfering with DNA replication. We hypothesized that the excess ß clamp sequesters the replicative DNA polymerase III (Pol III) to inhibit replication. As a test of this hypothesis, we obtained eight mutant clamps with an inability to impede growth and measured their ability to stimulate Pol III replication in vitro. Compared with the wild-type clamp, seven of the mutants were defective, consistent with their elevated cellular levels failing to sequester Pol III. However, the ßE202K mutant that bears a glutamic acid-to-lysine substitution at residue 202 displayed an increased affinity for Pol IIIα and Pol III core (Pol IIIαεθ), suggesting that it could still sequester Pol III effectively. Of interest, ßE202K supported in vitro DNA replication by Pol II and Pol IV but was defective with Pol III. Genetic experiments indicated that the dnaNE202K strain remained proficient in DNA damage-induced mutagenesis but was induced modestly for SOS and displayed sensitivity to UV light and methyl methanesulfonate. These results correlate an impaired ability of the mutant ßE202K clamp to support Pol III replication in vivo with its in vitro defect in DNA replication. Taken together, our results (i) support the model that sequestration of Pol III contributes to growth inhibition, (ii) argue for the existence of an additional mechanism that contributes to lethality, and (iii) suggest that physical and functional interactions of the ß clamp with Pol III are more extensive than appreciated currently. IMPORTANCE The ß clamp plays critically important roles in managing the actions of multiple proteins at the replication fork. However, we lack a molecular understanding of both how the clamp interacts with these different partners and the mechanisms by which it manages their respective actions. We previously exploited the finding that an elevated cellular level of the ß clamp impedes Escherichia coli growth by interfering with DNA replication. Using a genetic selection method, we obtained novel mutant ß clamps that fail to inhibit growth. Their analysis revealed that ßE202K is unique among them. Our work offers new insights into how the ß clamp interacts with and manages the actions of E. coli DNA polymerases II, III, and IV.

Elevated Levels of the Escherichia coli nrdAB-Encoded Ribonucleotide Reductase Counteract the Toxicity Caused by an Increased Abundance of the ß Clamp.

Expression of the Escherichia coli dnaN-encoded ß clamp at ≥10-fold higher than chromosomally expressed levels impedes growth by interfering with DNA replication. A mutant clamp (ßE202K bearing a glutamic acid-to-lysine substitution at residue 202) binds to DNA polymerase III (Pol III) with higher affinity than the wild-type clamp, suggesting that its failure to impede growth is independent of its ability to sequester Pol III away from the replication fork. Our results demonstrate that the dnaNE202K strain underinitiates DNA replication due to insufficient levels of DnaA-ATP and expresses several DnaA-regulated genes at altered levels, including nrdAB, that encode the class 1a ribonucleotide reductase (RNR). Elevated expression of nrdAB was dependent on hda function. As the ß clamp-Hda complex regulates the activity of DnaA by stimulating its intrinsic ATPase activity, this finding suggests that the dnaNE202K allele supports an elevated level of Hda activity in vivo compared with the wild-type strain. In contrast, using an in vitro assay reconstituted with purified components the ßE202K and wild-type clamp proteins supported comparable levels of Hda activity. Nevertheless, co-overexpression of the nrdAB-encoded RNR relieved the growth defect caused by elevated levels of the ß clamp. These results support a model in which increased cellular levels of DNA precursors relieve the ability of elevated ß clamp levels to impede growth and suggest either that multiple effects stemming from the dnaNE202K mutation contribute to elevated nrdAB levels or that Hda plays a noncatalytic role in regulating DnaA-ATP by sequestering it to reduce its availability. IMPORTANCE DnaA bound to ATP acts in initiation of DNA replication and regulates the expression of several genes whose products act in DNA metabolism. The state of the ATP bound to DnaA is regulated in part by the ß clamp-Hda complex. The dnaNE202K allele was identified by virtue of its inability to impede growth when expressed ≥10-fold higher than chromosomally expressed levels. While the dnaNE202K strain exhibits several phenotypes consistent with heightened Hda activity, the wild-type and ßE202K clamp proteins support equivalent levels of Hda activity in vitro. Taken together, these results suggest that ßE202K-Hda plays a noncatalytic role in regulating DnaA-ATP. This, as well as alternative models, is discussed.

Degradation of the Escherichia coli Essential Proteins DapB and Dxr Results in Oxidative Stress, which Contributes to Lethality through Incomplete Base Excision Repair.

Various lethal stresses, including bactericidal antibiotics, can trigger the production of reactive oxygen species (ROS) that contribute to killing. Incomplete base excision repair (BER) of oxidized nucleotides, especially 8-oxo-dG, has been identified as a major component of ROS-induced lethality. However, the relative contributions of this pathway to death vary widely between stresses, due in part to poorly understood complex differences in the physiological changes caused by these stresses. To identify new lethal stresses that kill cells through this pathway, we screened an essential protein degradation library and found that depletion of either DapB or Dxr leads to cell death through incomplete BER; the contribution of this pathway to overall cell death is greater for DapB than for Dxr. Depletion of either protein generates oxidative stress, which increases incorporation of 8-oxo-dG into the genome. This oxidative stress is causally related to cell death, as plating on an antioxidant provided a protective effect. Moreover, incomplete BER was central to this cell death, as mutants lacking the key BER DNA glycosylases MutM and MutY were less susceptible, while overexpression of the nucleotide sanitizer MutT, which degrades 8-oxo-dGTP to prevent its incorporation, was protective. RNA sequencing of cells depleted of these proteins revealed widely different transcriptional responses to these stresses. Our discovery that oxidative stress-induced incomplete BER is highly dependent on the exact physiological changes that the cell experiences helps explain the past confusion that arose concerning the role of ROS in antibiotic lethality. IMPORTANCE Bacterial cell death is a poorly understood process. The generation of reactive oxygen species (ROS) is an apparently common response to challenges by a wide variety of lethal stresses, including bactericidal antibiotics. Incomplete BER of nucleotides damaged by these ROS, especially 8-oxo-dG, is a significant contributing factor to this lethality, but the levels of its contribution vary widely between different lethal stresses. A better understanding of the conditions that cause cells to die because of incomplete BER may lead to improved strategies for targeting this mode of death as an adjunct to antimicrobial therapy.

Outbreak of ceftriaxone-resistant Salmonella enterica serotype Typhi-Tiruchirappalli, Tamil Nadu, India, June 2018.

Objectives: In May 2018, a laboratory network for antimicrobial resistance (AMR) surveillance in Tamil Nadu, India, detected a cluster of Salmonella enterica serotype Typhi (S. Typhi) isolates resistant to ceftriaxone. We investigated to describe the epidemiology and identify risk factors for the outbreak. Methods: We conducted unmatched case-control studies. We defined a case as illness (fever with abdominal pain, diarrhea or vomiting) in a person with blood culture-confirmed ceftriaxone-resistant S. Typhi isolated between January 1 and July 4, 2018 in Tiruchirappalli, Tamil Nadu. We interviewed cases using a semi-structured questionnaire to identify common exposures to food, water and places visited. Results: We identified 7 cases (5 men) during March 25-June 8, 2018, median age 23 years (range: 12-42); all were hospitalized, none died. Eating at Restaurant A (odds ratio [OR]=22) and chicken gravy (OR=16) was associated with illness. Of the 10 workers at Restaurant A, stool culture from 8 did not detect S. Typhi; 2 did not consent to provide samples. Five water samples around the restaurant showed low or no residual chlorine content. Conclusions: The investigation highlights the value of AMR surveillance in detecting emerging pathogens and the need for timely investigations, along with strengthening food safety.

Sinorhizobium meliloti YbeY is a zinc-dependent single-strand specific endoribonuclease that plays an important role in 16S ribosomal RNA processing.

Single-strand specific endoribonuclease YbeY has been shown to play an important role in the processing of the 3' end of the 16S rRNA in Escherichia coli. Lack of YbeY results in the accumulation of the 17S rRNA precursor. In contrast to a previous report, we show that Sinorhizobium meliloti YbeY exhibits endoribonuclease activity on single-stranded RNA substrate but not on the double-stranded substrate. This study also identifies the previously unknown metal ion involved in YbeY function to be Zn2+ and shows that the activity of YbeY is enhanced when the occupancy of zinc is increased. We have identified a pre-16S rRNA precursor that accumulates in the S. meliloti ΔybeY strain. We also show that ΔybeY mutant of Brucella abortus, a mammalian pathogen, also accumulates a similar pre-16S rRNA. The pre-16S species is longer in alpha-proteobacteria than in gamma-proteobacteria. We demonstrate that the YbeY from E. coli and S. meliloti can reciprocally complement the rRNA processing defect in a ΔybeY mutant of the other organism. These results establish YbeY as a zinc-dependent single-strand specific endoribonuclease that functions in 16S rRNA processing in both alpha- and gamma-proteobacteria.

Proteome Based de novo Sequencing of Novel Conotoxins from Marine Molluscivorous Cone Snail Conus amadis and Neurological Activities of Its Natural Venom in Zebrafish Model.

BACKGROUND: Conus amadis is a carnivorous snail found abundantly in coastal waters of India. Despite its abundance in southern coastal waters of India and the fact that most of the conotoxin act in neuronal system, research work on Conus amadis venom was not much focused. So we have made a brief study on the venom complex of Conus amadis to identify the library of novel conotoxins and to screen the natural venom for neurological function. OBJECTIVE: De novo sequencing of novel conopeptides from the venom cocktail of Conus amadis and to screen its natural venom for the presence of biological activities in zebrafish model. METHODS: Proteome based MALDI-TOF and LC-MS-MS analysis for identification of novel conotoxins and subsequent sequencing. Due to the complex disulfide rich nature of the venom peptides, the study also involves global chemical modification experiments of the venom extract to unambiguously determine the sequence of novel conotoxins. Biological function analysis of natural venom was tested in zebrafish model to ascertain anti-epileptic properties. RESULTS: In this study, we have identified 19 novel conotoxins containing 1, 2 & 3 disulfides, belonging to different classes. Among them, 2 novel contryphans, 3 T-superfamily conotoxins, 2 A-superfamily conotoxins and 2 Mini M-Superfamily conotoxins were sequenced to its amino acid level from the fragmented spectrum of singly and doubly charged parent ions using de novo sequencing strategies. ama1054, a contryphan peptide toxin, possesses post translationally modified bromo tryptophan at its seventh position. Except ama1251, all the sequenced peptide toxins possess modified C-terminal amidation. Crude venom exhibited anticonvulsant properties in pentylenetetrazole-induced seizure in zebrafish larvae, which suggested anti-epileptic property of the venom cocktail. Acetylcholinesterase activity was also identified in the venom complex. CONCLUSION: Based on the preliminary evidence, if this study is extended further through bioassay guided purification, could possibly yield peptide toxins with anticonvulsant and other neurologically active molecules.

Elevated Levels of Era GTPase Improve Growth, 16S rRNA Processing, and 70S Ribosome Assembly of Escherichia coli Lacking Highly Conserved Multifunctional YbeY Endoribonuclease.

YbeY is a highly conserved, multifunctional endoribonuclease that plays a significant role in ribosome biogenesis and has several additional roles. Here we show that overexpression of the conserved GTPase Era in Escherichia coli partially suppresses the growth defect of a ΔybeY strain while improving 16S rRNA processing and 70S ribosome assembly. This suppression requires both the ability of Era to hydrolyze GTP and the function of three exoribonucleases, RNase II, RNase R, and RNase PH, suggesting a model for the action of Era. Overexpression of Vibrio cholerae Era similarly partially suppresses the defects of an E. coli ΔybeY strain, indicating that this property of Era is conserved in bacteria other than E. coliIMPORTANCE This work provides insight into the critical, but still incompletely understood, mechanism of processing of the E. coli 16S rRNA 3' terminus. The highly conserved GTPase Era is known to bind to the precursor of the 16S rRNA near its 3' end. Both the endoribonuclease YbeY, which binds to Era, and four exoribonucleases have been implicated in this 3'-end processing. The results reported here offer additional insights into the role of Era in 16S rRNA 3'-end maturation and into the relationship between the action of the endoribonuclease YbeY and that of the four exoribonucleases. This study also hints at why YbeY is essential only in some bacteria and suggests that YbeY could be a target for a new class of antibiotics in these bacteria.

Dynamic assembly of Hda and the sliding clamp in the regulation of replication licensing.

Regulatory inactivation of DnaA (RIDA) is one of the major regulatory mechanisms of prokaryotic replication licensing. In RIDA, the Hda-sliding clamp complex loaded onto DNA directly interacts with adenosine triphosphate (ATP)-bound DnaA and stimulates the hydrolysis of ATP to inactivate DnaA. A prediction is that the activity of Hda is tightly controlled to ensure that replication initiation occurs only once per cell cycle. Here, we determined the crystal structure of the Hda-ß clamp complex. This complex contains two pairs of Hda dimers sandwiched between two ß clamp rings to form an octamer that is stabilized by three discrete interfaces. Two separate surfaces of Hda make contact with the ß clamp, which is essential for Hda function in RIDA. The third interface between Hda monomers occludes the active site arginine finger, blocking its access to DnaA. Taken together, our structural and mutational analyses of the Hda-ß clamp complex indicate that the interaction of the ß clamp with Hda controls the ability of Hda to interact with DnaA. In the octameric Hda-ß clamp complex, the inability of Hda to interact with DnaA is a novel mechanism that may regulate Hda function.

C21orf57 is a human homologue of bacterial YbeY proteins.

The product of the human C21orf57 (huYBEY) gene is predicted to be a homologue of the highly conserved YbeY proteins found in nearly all bacteria. We show that, like its bacterial and chloroplast counterparts, the HuYbeY protein is an RNase and that it retains sufficient function in common with bacterial YbeY proteins to partially suppress numerous aspects of the complex phenotype of an Escherichia coli ΔybeY mutant. Expression of HuYbeY in Saccharomyces cerevisiae, which lacks a YbeY homologue, results in a severe growth phenotype. This observation suggests that the function of HuYbeY in human cells is likely regulated through specific interactions with partner proteins similarly to the way YbeY is regulated in bacteria.

Insufficient levels of the nrdAB-encoded ribonucleotide reductase underlie the severe growth defect of the Δhda E. coli strain.

The ATP-bound form of the Escherichia coli DnaA replication initiator protein remodels the chromosomal origin of replication, oriC, to load the replicative helicase. The primary mechanism for regulating the activity of DnaA involves the Hda and ß clamp proteins, which act together to dramatically stimulate the intrinsic DNA-dependent ATPase activity of DnaA via a process termed Regulatory Inactivation of DnaA. In addition to hyperinitiation, strains lacking hda function also exhibit cold sensitive growth at 30°C. Strains impaired for the other regulators of initiation (i.e., ΔseqA or ΔdatA) fail to exhibit cold sensitivity. The goal of this study was to gain insight into why loss of hda function impedes growth. We used a genetic approach to isolate 9 suppressors of Δhda cold sensitivity, and characterized the mechanistic basis by which these suppressors alleviated Δhda cold sensitivity. Taken together, our results provide strong support for the view that the fundamental defect associated with Δhda is diminished levels of DNA precursors, particularly dGTP and dATP. We discuss possible mechanisms by which the suppressors identified here may regulate dNTP pool size, as well as similarities in phenotypes between the Δhda strain and hda+ strains exposed to the ribonucleotide reductase inhibitor hydroxyurea.

Identification of ß Clamp-DNA Interaction Regions That Impair the Ability of E. coli to Tolerate Specific Classes of DNA Damage.

The E. coli dnaN-encoded ß sliding clamp protein plays a pivotal role in managing the actions on DNA of the 5 bacterial DNA polymerases, proteins involved in mismatch repair, as well as several additional proteins involved in DNA replication. Results of in vitro experiments indicate that the loading of ß clamp onto DNA relies on both the DnaX clamp loader complex as well as several discrete sliding clamp-DNA interactions. However, the importance of these DNA interactions to E. coli viability, as well as the ability of the ß clamp to support the actions of its numerous partner proteins, have not yet been examined. To determine the contribution of ß clamp-DNA interactions to the ability of E. coli to cope with different classes of DNA damage, we used alanine scanning to mutate 22 separate residues mapping to 3 distinct ß clamp surfaces known or nearby those known to contact the DNA template, including residues P20-L27 (referred to here as loop I), H148-Y154 (loop II) and 7 different residues lining the central pore of the ß clamp through which the DNA template threads. Twenty of these 22 dnaN mutants supported bacterial growth. While none of these 20 conferred sensitivity to hydrogen peroxide or ultra violet light, 12 were sensitized to NFZ, 5 were sensitized to MMS, 8 displayed modestly altered frequencies of DNA damage-induced mutagenesis, and 2 may be impaired for supporting hda function. Taken together, these results demonstrate that discrete ß clamp-DNA interaction regions contribute to the ability of E. coli to tolerate specific classes of DNA damage.

The sliding clamp tethers the endonuclease domain of MutL to DNA.

The sliding clamp enhances polymerase processivity and coordinates DNA replication with other critical DNA processing events including translesion synthesis, Okazaki fragment maturation and DNA repair. The relative binding affinity of the sliding clamp for its partners determines how these processes are orchestrated and is essential to ensure the correct processing of newly replicated DNA. However, while stable clamp interactions have been extensively studied; dynamic interactions mediated by the sliding clamp remain poorly understood. Here, we characterize the interaction between the bacterial sliding clamp (ß-clamp) and one of its weak-binding partners, the DNA mismatch repair protein MutL. Disruption of this interaction causes a mild mutator phenotype in Escherichia coli, but completely abrogates mismatch repair activity in Bacillus subtilis. We stabilize the MutL-ß interaction by engineering two cysteine residues at variable positions of the interface. Using disulfide bridge crosslinking, we have stabilized the E. coli and B. subtilis MutL-ß complexes and have characterized their structures using small angle X-ray scattering. We find that the MutL-ß interaction greatly stimulates the endonuclease activity of B. subtilis MutL and supports this activity even in the absence of the N-terminal region of the protein.

A dnaN plasmid shuffle strain for rapid in vivo analysis of mutant Escherichia coli ß clamps provides insight into the role of clamp in umuDC-mediated cold sensitivity.

The E. coli umuDC gene products participate in two temporally distinct roles: UmuD2C acts in a DNA damage checkpoint control, while UmuD'2C, also known as DNA polymerase V (Pol V), catalyzes replication past DNA lesions via a process termed translesion DNA synthesis. These different roles of the umuDC gene products are managed in part by the dnaN-encoded ß sliding clamp protein. Co-overexpression of the ß clamp and Pol V severely blocked E. coli growth at 30°C. We previously used a genetic assay that was independent of the ability of ß clamp to support E. coli viability to isolate 8 mutant clamp proteins (ßQ61K, ßS107L, ßD150N, ßG157S, ßV170M, ßE202K, ßM204K and ßP363S) that failed to block growth at 30°C when co-overexpressed with Pol V. It was unknown whether these mutant clamps were capable of supporting E. coli viability and normal umuDC functions in vivo. The goals of this study were to answer these questions. To this end, we developed a novel dnaN plasmid shuffle assay. Using this assay, ßD150N and ßP363S were unable to support E. coli viability. The remaining 6 mutant clamps, each of which supported viability, were indistinguishable from ß+ with respect to umuDC functions in vivo. In light of these findings, we analyzed phenotypes of strains overexpressing either ß clamp or Pol V alone. The strain overexpressing ß+, but not those expressing mutant ß clamps, displayed slowed growth irrespective of the incubation temperature. Moreover, growth of the Pol V-expressing strain was modestly slowed at 30°, but not 42°C. Taken together, these results suggest the mutant clamps were identified due to their inability to slow growth rather than an inability to interact with Pol V. They further suggest that cold sensitivity is due, at least in part, to the combination of their individual effects on growth at 30°C.