A Novel Device for the Evaluation of In Vitro Bacterial Colonization in Membranes for Guided Tissue and Bone Regeneration.

Purpose: To evaluate, in vitro, the efficiency of a novel apparatus to test the adherence and penetration of bacteria on different membranes for guided regeneration. Methodology: To create the 3D device, Computer Aided Design/Computer Aided Manufacturing (CAD/CAM) systems were used. Three types of biomaterials were tested (n = 6): (DT) a collagen membrane; (DS) a polymer membrane; and (LP) a dense polytetrafluoroethylene barrier. The biomaterials were adapted to the apparatuses and challenged with two different monospecies bacterial culture of A. actinomycetemcomitans b and S. mutans. After 2 h, bacterial adherence and penetration were quantified by counting the number of colony-forming units (CFUs). Two specimens from each group were used for image analysis using Confocal Laser Scanning Microscopy. Statistical analysis was performed. Findings: The DS group had a higher adherence of S. mutans compared to A. actinomycetemcomitans b (p = 0.05). There was less adherence of A. actinomycetemcomitans b in the DS group, compared to the LP (p = 0.011) and DT (p < 0.001) groups. Only the membranes allowed penetration, which was blocked by barriers. The DT group allowed a greater penetration of S. mutans to occur compared to A. actinomycetemcomitans b (p = 0.009), which showed a higher penetration into the DS membranes compared to S. mutans (p = 0.016). The penetration of A. actinomycetemcomitans b through DS was higher compared to its penetration through DT and LP (p < 0.01 for both). DT and DS allowed a greater penetration of S. mutans to occur compared to LP, which prevented both bacterial species from penetrating. Conclusion: The apparatus allowed for the settlement and complete sealing of the biomaterials, enabling standardization.

Prevalence of species of yellow, purple and green microbial complexes in endo-perio lesions: a systematic review.

This review aimed to determine the prevalence of species of yellow, purple and green microbial complexes in root canals (RC) and periodontal pockets (PP) of teeth with endodontic-periodontal lesions. For this purpose, two reviewers searched the literature up to January 2022. Studies reporting the prevalence of species of the yellow, purple and green microbial complexes in teeth diagnosed with endodontic-periodontal lesions were included. The risk of bias of the included studies was assessed using the 14 criteria from the NIH Quality Assessment Tool. Of 1,611 references identified in the initial search, only four studies were eligible and included in the qualitative analysis. The profile and prevalence rates of bacterial species in RC and PP varied among the included studies: levels of Agregatibacter actinomycetemcomitans (12% RC, 58% PP), Capnocytophaga granulosa (10% RC, 35% PP), Capnocytophaga sputigena (15-70% RC, 0-30% PP), Streptococcus mitis (30% RC, 35% PP), Streptococcus sanguinis (30% RC, 35% PP), and Veillonella parvula (70% RC, 50% PP) were identified. The high methodological heterogeneity prevented grouping and quantitative analysis of data. The risk of bias was considered 'moderate' for all studies. The included studies identified the presence of seven bacterial species belonging to the yellow, purple, and green microbial complexes in RC and PP, but with different prevalence rates. Future clinical studies are encouraged to investigate the presence and role of these species in the occurrence and development of endodontic-periodontal lesions.

Assessment of multispecies biofilm growth on root canal dentin under different radiation therapy regimens.

OBJECTIVES: To assess the growth of a multispecies biofilm on root canal dentin under different radiotherapy regimens. MATERIALS AND METHODS: Sixty-three human root dentin cylinders were distributed into six groups. In three groups, no biofilm was formed (n = 3): NoRT) non-irradiated dentin; RT55) 55 Gy; and RT70) 70 Gy. In the other three groups (n = 18), a 21-day multispecies biofilm (Enterococcus faecalis, Streptococcus mutans, and Candida albicans) was formed in the canal: NoRT + Bio) non-irradiated + biofilm; RT55 + Bio) 55 Gy + biofilm; and RT70 + Bio) 70 Gy + biofilm. The biofilm was quantified (CFUs/mL). Biofilm microstructure was assessed under SEM. Microbial penetration into dentinal tubules was assessed under CLSM. For the biofilm biomass and dentin microhardness pre- and after biofilm growth assessments, 45 bovine dentin specimens were distributed into three groups (n = 15): NoRT) non-irradiated + biofilm; RT55 + Bio) 55 Gy + biofilm; and RT70 + Bio) 70 Gy + biofilm. RESULTS: Irradiated specimens (70 Gy) had higher quantity of microorganisms than non-irradiated (p = .010). There was gradual increase in biofilm biomass from non-irradiated to 55 Gy and 70 Gy (p < .001). Irradiated specimens had greater reduction in microhardness after biofilm growth. Irradiated dentin led to the growth of a more complex and irregular biofilm. There was microbial penetration into the dentinal tubules, regardless of the radiation regimen. CONCLUSION: Radiotherapy increased the number of microorganisms and biofilm biomass and reduced dentin microhardness. Microbial penetration into dentinal tubules was noticeable. CLINICAL RELEVANCE: Cumulative and potentially irreversible side effects of radiotherapy affect biofilm growth on root dentin. These changes could compromise the success of endodontic treatment in oncological patients undergoing head and neck radiotherapy.

Prevalence of species of yellow, purple and green microbial complexes in endo-perio lesions: a systematic review

Abstract This review aimed to determine the prevalence of species of yellow, purple and green microbial complexes in root canals (RC) and periodontal pockets (PP) of teeth with endodontic-periodontal lesions. For this purpose, two reviewers searched the literature up to January 2022. Studies reporting the prevalence of species of the yellow, purple and green microbial complexes in teeth diagnosed with endodontic-periodontal lesions were included. The risk of bias of the included studies was assessed using the 14 criteria from the NIH Quality Assessment Tool. Of 1,611 references identified in the initial search, only four studies were eligible and included in the qualitative analysis. The profile and prevalence rates of bacterial species in RC and PP varied among the included studies: levels of Agregatibacter actinomycetemcomitans (12% RC, 58% PP), Capnocytophaga granulosa (10% RC, 35% PP), Capnocytophaga sputigena (15-70% RC, 0-30% PP), Streptococcus mitis (30% RC, 35% PP), Streptococcus sanguinis (30% RC, 35% PP), and Veillonella parvula (70% RC, 50% PP) were identified. The high methodological heterogeneity prevented grouping and quantitative analysis of data. The risk of bias was considered 'moderate' for all studies. The included studies identified the presence of seven bacterial species belonging to the yellow, purple, and green microbial complexes in RC and PP, but with different prevalence rates. Future clinical studies are encouraged to investigate the presence and role of these species in the occurrence and development of endodontic-periodontal lesions.

Candida albicans adhesion on 3D-printed and thermopolymerizable polymethyl methacrylate for removable prostheses.

PURPOSE: To compare Candida albicans adhesion and quantify the biofilm formed on thermopolymerizable (T.PMMA) and 3D printing (3D.PMMA) polymethyl methacrylate through quantitative and qualitative analyses. METHODS: The specimens were made (n= 39/material) by conventional polymerization (T.PMMA) and designed in Autodesk Meshmixer software, followed by 3D printing (3D.PMMA) - CAD-CAM method. After the roughness standardization, the monospecies biofilm of C. albicans (ATCC 10231) was submitted to adhesion analysis, verifying the quantification and microbial load, in triplicate, by the methods of counting colony forming units (CFU/mL) and staining with crystal violet, followed by optical density reading, respectively. Qualitative analyses were performed by scanning electron microscopy to analyze the surface characteristics of the specimens and biofilm formed. The Mann-Whitney U test was used for comparisons between resins regarding the count of CFU/mL (log¹°+ 1) and optical density values (P≤ 0.05). RESULTS: T.PMMA (5.78 ± 0.43) had a CFU/mL count (P< 0.001) higher than 3D.PMMA (4.84 ± 0.39). There was a difference between C. albicans biofilm accumulation (P< 0.001), in which on T.PMMA (1.59 ± 0.31) was higher than 3D.PMMA (1.10 ± 0.14). The qualitative analyses corresponded to the quantitative ones and allowed the visualization of more porosities in T.PMMA. CLINICAL SIGNIFICANCE: The adhesion (microbial load and biofilm accumulation) of C. albicans was lower in the 3D printed polymethyl methacrylate when compared to the thermopolymerizable model.

Oleate Hydratase in Lactobacillus delbrueckii subsp. bulgaricus LBP UFSC 2230 Catalyzes the Reversible Conversion between Linoleic Acid and Ricinoleic Acid.

Conjugated linoleic acid (CLA) has been the subject of numerous studies in recent decades because of its associated health benefits. CLA is an intermediate product of the biohydrogenation pathway of linoleic acid (LA) in bacteria. Several bacterial species capable of efficiently converting LA into CLA have been widely reported in the literature, among them Lactobacillus delbrueckii subsp. bulgaricus LBP UFSC 2230. Over the last few years, a multicomponent enzymatic system consisting of three enzymes involved in the biohydrogenation process of LA has been proposed. Sequencing the genome of L. delbrueckii subsp. bulgaricus LBP UFSC 2230 revealed only one gene capable of encoding an oleate hydratase (OleH), unlike the presence of multiple genes typically found in similar strains. This study investigated the biological effect of the OleH enzyme of L. delbrueckii subsp. bulgaricus LBP UFSC 2230 on the hydration of LA and dehydration of ricinoleic acid (RA) and its possible role in the production of CLA. The OleH was cloned, expressed, purified, and characterized. Fatty acid measurements were made by an internal standard method using a gas chromatography-coupled flame ionization detector (GC-FID) system. It was found that the enzyme is a hydratase/dehydratase, leading to a reversible transformation between LA and RA. In addition, the results showed that L. delbrueckii subsp. bulgaricus LBP UFSC 2230 OleH protein plays a role in stress tolerance in Escherichia coli. In conclusion, the OleH of L. delbrueckii subsp. bulgaricus LBP UFSC 2230 catalyzes the initial stage of saturation metabolism of LA, although it has not converted the substrates directly into CLA. IMPORTANCE This study provides insight into the enzymatic mechanism of CLA synthesis in L. delbrueckii subsp. bulgaricus and broadens our understanding of the bioconversion of LA and RA by OleH. The impact of OleH on the production of the c9, t11 CLA isomer and stress tolerance by E. coli has been assisted. The results provide an understanding of the factors which influence OleH activity. L. delbrueckii subsp. bulgaricus LBP UFSC 2230 OleH presented two putative fatty acid-binding sites. Recombinant OleH catalyzed both LA hydration and RA dehydration. OleH was shown to play a role in bacterial growth performance in the presence of LA.

Prevalence of red and orange microbial complexes in endodontic-periodontal lesions: a systematic review and meta-analysis.

OBJECTIVE: This systematic review assesses the prevalence of microbial complexes in endodontic-periodontal lesion. MATERIALS AND METHODS: Nine databases were searched through August 2020. Experts were consulted to indicate additional studies. Studies were blindly selected by two reviewers based on pre-defined eligibility criteria. Studies that evaluated the prevalence of microbial orange and red complexes among patients with endodontic-periodontal lesion were considered eligible. Risk of bias was assessed using the Joanna Briggs Institute Critical Appraisal Checklist for Studies Reporting Prevalence Data. RESULTS: From 572 articles found on all databases, 11 clinical studies were finally included. The following microorganisms were investigated: P. gingivalis, T. forsythia, T. denticola, F. nucleatum, F. periodonticum, P. micra, P. intermedia, P. nigrescens, S. constellatus, C. gracilis, C. rectus, C. showae and E. nodatum. Considering the orange complex, P. micra, E. nodatum and S. constellatus were prevalent in both root canal and periodontal pockets. P. gingivalis and T. forsythia belonging to the red complex were prevalent only in periodontal pockets. The red complex microorganisms were not found very frequently in root canal. CONCLUSIONS: There is a similarity between the microbiome of root canal and periodontal pockets, with prevalence of the three microorganisms of the orange complex. Two microorganisms from the red complex were prevalent only in periodontal pockets. CLINICAL RELEVANCE: The prevalence of specific microorganisms in endodontic-periodontal lesion is important to understand the microbiological profile of the patients involved and to correlate it with possible clinical and repair conditions of this pathology.

Draft genome sequence of Lactobacillus delbrueckii subsp. bulgaricus LBP UFSC 2230: a tool for preliminary identification of enzymes involved in CLA metabolism.

Several Lactobacillus ssp. are recognized as potential conjugated linoleic acid (CLA) producers. We have previously reported the ability of a range of Lactobacillus delbrueckii subsp. bulgaricus strains to produce CLA in fermented milk, being a potential candidate for the fermented dairy food chain. This study reports the draft genome sequence of L. bulgaricus strain LBP UFSC 2230, isolated from Italian Grana Padano cheese. Draft genome sequence originated in a total of 4,310,842 paired-end reads that were quality trimmed and assembled into 135 contigs with a total length of 604,745,873 bp, including 2086 protein coding genes and an average GC content of 49.7%. Draft genome sequence represents an important tool to identify the enzymes involved in this strain's CLA metabolism. We identified a gene encoding an enzyme involved in biohydrogenation of linoleic acid pathway, oleate hydratase.

Association of the Cold Shock DEAD-Box RNA Helicase RhlE to the RNA Degradosome in Caulobacter crescentus.

In diverse bacterial lineages, multienzyme assemblies have evolved that are central elements of RNA metabolism and RNA-mediated regulation. The aquatic Gram-negative bacterium Caulobacter crescentus, which has been a model system for studying the bacterial cell cycle, has an RNA degradosome assembly that is formed by the endoribonuclease RNase E and includes the DEAD-box RNA helicase RhlB. Immunoprecipitations of extracts from cells expressing an epitope-tagged RNase E reveal that RhlE, another member of the DEAD-box helicase family, associates with the degradosome at temperatures below those optimum for growth. Phenotype analyses of rhlE, rhlB, and rhlE rhlB mutant strains show that RhlE is important for cell fitness at low temperature and its role may not be substituted by RhlB. Transcriptional and translational fusions of rhlE to the lacZ reporter gene and immunoblot analysis of an epitope-tagged RhlE indicate that its expression is induced upon temperature decrease, mainly through posttranscriptional regulation. RNase E pulldown assays show that other proteins, including the transcription termination factor Rho, a second DEAD-box RNA helicase, and ribosomal protein S1, also associate with the degradosome at low temperature. The results suggest that the RNA degradosome assembly can be remodeled with environmental change to alter its repertoire of helicases and other accessory proteins.IMPORTANCE DEAD-box RNA helicases are often present in the RNA degradosome complex, helping unwind secondary structures to facilitate degradation. Caulobacter crescentus is an interesting organism to investigate degradosome remodeling with change in temperature, because it thrives in freshwater bodies and withstands low temperature. In this study, we show that at low temperature, the cold-induced DEAD-box RNA helicase RhlE is recruited to the RNA degradosome, along with other helicases and the Rho protein. RhlE is essential for bacterial fitness at low temperature, and its function may not be complemented by RhlB, although RhlE is able to complement for rhlB loss. These results suggest that RhlE has a specific role in the degradosome at low temperature, potentially improving adaptation to this condition.

Role and regulation of ferritin-like proteins in iron homeostasis and oxidative stress survival of Caulobacter crescentus.

Iron is an essential nutrient that is poorly available to living organisms but can be harmful when in excess due to the production of reactive oxygen species. Bacteria and other organisms use iron storage proteins called ferritins to avoid iron toxicity and as a safe iron source in the cytosol. The alpha-proteobacterium Caulobacter crescentus has two putative ferritins, Bfr and Dps, and some other proteins belonging to the ferritin-like superfamily, among them the one encoded by CC_0557. In this work, we have analyzed the role and regulation of these three putative ferritin-like proteins. Using lacZ-transcriptional fusions, we found that bfr expression is positively regulated (2.5-fold induction) by the Fe-responsive regulator Fur in iron sufficiency, as expected for an iron storage protein. Expression of dps was induced 1.5-fold in iron limitation in a Fur-independent manner, while the expression of the product of CC_0557 was unaffected by either iron supply or Fur. With respect to growth phase, while bfr expression was constant during growth, expression of dps (1.4-fold) and CC_0557 (around seven times) increased in the transition from exponential to stationary phase. Deletion mutant strains for each gene and a double dps/bfr mutant were obtained and tested for oxidative stress resistance. The dps mutant was very sensitive to H2O2, and this phenotype was not relieved by the addition of the iron chelator 2',2-dipyridyl in the conditions tested. While bfr and CC_0557 showed no phenotype as to H2O2 resistance, the double dps/bfr mutant had a similar phenotype to the dps mutation alone. These findings indicate that in C. crescentus Bfr contributes to iron homeostasis and Dps has a role in protection against oxidative stress. The role of the protein CC_0557 containing a ferritin-like fold remains unclear.

Transcriptomic analysis of the stationary phase response regulator SpdR in Caulobacter crescentus.

BACKGROUND: As bacterial cells enter stationary phase, they adjust their growth rate to comply with nutrient restriction and acquire increased resistance to several stresses. These events are regulated by controlling gene expression at this phase, changing the mode of exponential growth into that of growth arrest, and increasing the expression of proteins involved in stress resistance. The two-component system SpdR/SpdS is required for the activation of transcription of the Caulobacter crescentus cspD gene at the onset of stationary phase. RESULTS: In this work, we showed that both SpdR and SpdS are also induced upon entry into stationary phase, and this induction is partly mediated by ppGpp and it is not auto-regulated. Global transcriptional analysis at early stationary phase of a spdR null mutant strain compared to the wild type strain was carried out by DNA microarray. Twenty-three genes showed at least twofold decreased expression in the spdR deletion mutant strain relative to its parental strain, including cspD, while five genes showed increased expression in the mutant. The expression of a set of nine genes was evaluated by quantitative real time PCR, validating the microarray data, and indicating an important role for SpdR at stationary phase. Several of the differentially expressed genes can be involved in modulating gene expression, including four transcriptional regulators, and the RNA regulatory protein Hfq. The ribosomal proteins NusE and NusG, which also have additional regulatory functions in transcription and translation, were also downregulated in the spdR mutant, as well as the ParE1 toxin. The purified SpdR protein was shown to bind to the regulatory region of CC0517 by Electrophoretic Mobility Shift Assay, and the SpdR-regulated gene CC0731 was shown to be expressed at a lower level in the null cspD mutant, suggesting that at least part of the effect of SpdR on the expression of this gene is indirect. CONCLUSIONS: The results indicate that SpdR regulates several genes encoding proteins of regulatory function, which in turn may be required for the expression of other genes important for the transition to stationary phase.

Analysis of the Caulobacter crescentus Zur regulon reveals novel insights in zinc acquisition by TonB-dependent outer membrane proteins.

BACKGROUND: Intracellular zinc concentration needs to be maintained within strict limits due to its toxicity at high levels, and this is achieved by a finely regulated balance between uptake and efflux. Many bacteria use the Zinc Uptake Regulator Zur to orchestrate zinc homeostasis, but little is known regarding the transport of this metal across the bacterial outer membrane. RESULTS: In this work we determined the Caulobacter crescentus Zur regulon by global transcriptional and in silico analyses. Among the genes directly repressed by Zur in response to zinc availability are those encoding a putative high affinity ABC uptake system (znuGHI), three TonB-dependent receptors (znuK, znuL and znuM) and one new putative transporter of a family not yet characterized (zrpW). Zur is also directly involved in the activation of a RND and a P-type ATPase efflux systems, as revealed by ß-galactosidase and site-directed mutagenesis assays. Several genes belonging to the Fur regulon were also downregulated in the zur mutant, suggesting a putative cross-talk between Zur and Fur regulatory networks. Interestingly, a phenotypic analysis of the znuK and znuL mutants has shown that these genes are essential for growth under zinc starvation, suggesting that C. crescentus uses these TonB-dependent outer membrane transporters as key zinc scavenging systems. CONCLUSIONS: The characterization of the C. crescentus Zur regulon showed that this regulator coordinates not only uptake, but also the extrusion of zinc. The uptake of zinc by C. crescentus in conditions of scarcity of this metal is highly dependent on TonB-dependent receptors, and the extrusion is mediated by an RND and P-type ATPase transport systems. The absence of Zur causes a disturbance in the dynamic equilibrium of zinc intracellular concentration, which in turn can interfere with other regulatory networks as seen for the Fur regulon.

Cold shock genes cspA and cspB from Caulobacter crescentus are posttranscriptionally regulated and important for cold adaptation.

Cold shock proteins (CSPs) are nucleic acid binding chaperones, first described as being induced to solve the problem of mRNA stabilization after temperature downshift. Caulobacter crescentus has four CSPs: CspA and CspB, which are cold induced, and CspC and CspD, which are induced only in stationary phase. In this work we have determined that the synthesis of both CspA and CspB reaches the maximum levels early in the acclimation phase. The deletion of cspA causes a decrease in growth at low temperature, whereas the strain with a deletion of cspB has a very subtle and transient cold-related growth phenotype. The cspA cspB double mutant has a slightly more severe phenotype than that of the cspA mutant, suggesting that although CspA may be more important to cold adaptation than CspB, both proteins have a role in this process. Gene expression analyses were carried out using cspA and cspB regulatory fusions to the lacZ reporter gene and showed that both genes are regulated at the transcriptional and posttranscriptional levels. Deletion mapping of the long 5'-untranslated region (5'-UTR) of each gene identified a common region important for cold induction, probably via translation enhancement. In contrast to what was reported for other bacteria, these cold shock genes have no regulatory regions downstream from ATG that are important for cold induction. This work shows that the importance of CspA and CspB to C. crescentus cold adaptation, mechanisms of regulation, and pattern of expression during the acclimation phase apparently differs in many aspects from what has been described so far for other bacteria.

SpdR, a response regulator required for stationary-phase induction of Caulobacter crescentus cspD.

The cold shock protein (CSP) family includes small polypeptides that are induced upon temperature downshift and stationary phase. The genome of the alphaproteobacterium Caulobacter crescentus encodes four CSPs, with two being induced by cold shock and two at the onset of stationary phase. In order to identify the environmental signals and cell factors that are involved in cspD expression at stationary phase, we have analyzed cspD transcription during growth under several nutrient conditions. The results showed that expression of cspD was affected by the medium composition and was inversely proportional to the growth rate. The maximum levels of expression were decreased in a spoT mutant, indicating that ppGpp may be involved in the signalization for carbon starvation induction of cspD. A Tn5 mutant library was screened for mutants with reduced cspD expression, and 10 clones that showed at least a 50% reduction in expression were identified. Among these, a strain with a transposon insertion into a response regulator of a two-component system showed no induction of cspD at stationary phase. This protein (SpdR) was able to acquire a phosphate group from its cognate histidine kinase, and gel mobility shift assay and DNase I footprinting experiments showed that it binds to an inverted repeat sequence of the cspD regulatory region. A mutated SpdR with a substitution of the conserved aspartyl residue that is the probable phosphorylation site is unable to bind to the cspD regulatory region and to complement the spdR mutant phenotype.

CspC and CspD are essential for Caulobacter crescentus stationary phase survival.

The cold shock response in bacteria involves the expression of low-molecular weight cold shock proteins (CSPs) containing a nucleic acid-binding cold shock domain (CSD), which are known to destabilize secondary structures on mRNAs, facilitating translation at low temperatures. Caulobacter crescentus cspA and cspB are induced upon cold shock, while cspC and cspD are induced during stationary phase. In this work, we determined a new coding sequence for the cspC gene, revealing that it encodes a protein containing two CSDs. The phenotypes of C. crescentus csp mutants were analyzed, and we found that cspC is important for cells to maintain viability during extended periods in stationary phase. Also, cspC and cspCD strains presented altered morphology, with frequent non-viable filamentous cells, and cspCD also showed a pronounced cell death at late stationary phase. In contrast, the cspAB mutant presented increased viability in this phase, which is accompanied by an altered expression of both cspC and cspD, but the triple cspABD mutant loses this characteristic. Taken together, our results suggest that there is a hierarchy of importance among the csp genes regarding stationary phase viability, which is probably achieved by a fine tune balance of the levels of these proteins.

Characterization of Caulobacter crescentus response to low temperature and identification of genes involved in freezing resistance.

Free-living bacteria must respond to a wide range of temperature changes, and have developed specific mechanisms to survive in extreme environments. In this work we describe a remarkable resistance of mesophilic bacterium Caulobacter crescentus to several cycles of freezing at -80 degrees C, which was able to grow at low temperatures. Exponentially growing cells and late stationary-phase cells presented higher freezing resistance at both -20 and -80 degrees C than early stationary-phase cells. Cryotolerance was observed when log-phase cultures grown at 30 degrees C were preincubated at 5, 15 or 20 degrees C before freezing at -20 degrees C. A transposon library was screened to identify mutants sensitive to freezing at -80 degrees C and three strains presenting <10% survival were isolated. Identification of genes disrupted in each mutant showed that they encoded an AddA family DNA helicase, a DEAD/DEAH box RNA helicase and a putative RND (resistance, nodulation, cell division) efflux system component. These strains showed longer generation times than wild-type cells when growing at 15 degrees C, with the RNA helicase mutant presenting a severe growth defect. These analyses suggest that the singular intrinsic resistance to freezing of C. crescentus is in fact a consequence of several independent traits, especially the maintenance of a proper degree of supercoiling of nucleic acids.