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Transcriptional regulation of the deoP2 promoter by the cyclic AMP/cyclic AMP receptor protein complex (cAMP/CRP) and the CytR repressor requires two high-affinity CRP targets located around -41 and -93bp preceding the start site for transcription. Here we report the structure of cddP, another CRP/CytR-regulated promoter. In common with what was found in deo, the cdd promoter also contains multiple CRP targets. Thus, using the DNasel footprinting procedure, tandem CRP binding sites were identified around -41 and -93. These findings support a general model for CytR binding and CytR regulation, in which (i) CytR and the CRP/cAMP complex bind to similar or Identical targets, (ii) two or more targets are necessary for proper binding of CytR to a promoter region, and (iii) CytR represses transcription by antagonizing cAMP/CRP activation. [ABSTRACT FROM AUTHOR]

Objective: To assess the impact of NRP virtual simulations (eSim™), video or no refresher training, on simulation performance, six months after a provider course; and to evaluate eSim™ acceptability., Study Design: In this multi-site randomized controlled trial, NRP providers from four U.S. institutions were randomized to receive refreshers every two months with NRP eSim™, NRP resuscitation video, or no refresher (control). Simulation performance was assessed immediately after an NRP course and six months later., Result: 248 participants completed the baseline simulation and 148 completed the six-month follow-up simulation. The majority (71%) of subjects had a decline in resuscitation skills at 6 months. There were no differences in performance between the study groups, but participants who reported using either the video or eSim™ had less decline in performance at the 6-month follow-up (p < 0.05)., Conclusion: NRP refreshers with either eSim™ or NRP video may mitigate the decline in resuscitation skills after training., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Objective: Mothers of preterm infants face unique challenges in establishing milk supply. We hypothesized that daily meditation practice while pumping would increase milk volume., Study Design: This was a randomized control trial examining the effect of meditation on the breastmilk supply of women delivering preterm infants. The meditation group engaged in daily use of a mindfulness-focused meditation app in addition to routine lactation support. The primary outcome was mean breastmilk volume on the infant's nineth day of life. Secondary outcomes included use of lactation-promoting behaviors, continuation of breastfeeding, and measures of mental health and breastfeeding self-efficacy by validated questionnaires. In addition to the intention to treat analysis, a per protocol analysis examined the association of frequent meditation with these breastfeeding and mental health outcomes., Results: A total of 60 women were analyzed. Mean milk volume was 647.1 ± 467.8 mL in the meditation group and 514.9 ± 393.5 mL in the routine care group ( p  = 0.27). Median number of pumping sessions was 7 (interquartile range [IQR] 5-8) in the meditation group, compared to 6 (IQR 4-7) in the routine care group ( p  = 0.11). Other lactation-promoting behaviors, breastfeeding continuation, and questionnaire scores were similar.Adjusting for confounders, we found an increase in breastmilk production of 223.2 mL (95% CI 98.8-347.5, p  = 0.001) and in pumping episodes by 0.93 (95% CI 0.16-1.70, p  = 0.020) associated with frequent meditation. Skin-to-skin contact was increased to 100% ( p  = 0.006) among women who meditated seven or more times. Adjusted odds of a clinically significant Edinburgh Postnatal Depression Scale score of >9 was 0.057 (95% CI 0.0014-0.711, p  = 0.023) with frequent meditation., Conclusion: Breastmilk production was similar in mothers practicing meditation compared to those receiving routine lactation support. For women engaging in frequent mediation, there may be an effect in establishing breastmilk supply and reduction of depression symptoms., Key Points: · Breastmilk is not increased for neonatal intensive care unit (NICU) moms asked to engage in meditation.. · Maternal mental health is not improved among NICU moms asked to meditate.. · However, higher-frequency meditation is associated with increased milk volume and lower odds of high scores on depression screening.., Competing Interests: None declared., (Thieme. All rights reserved.)

Aim: Neonatal resuscitation guidelines promote the laryngeal mask (LM) interface for positive pressure ventilation (PPV), but little is known about how the LM is used among Neonatal Resuscitation Program (NRP) Providers and Instructors. The study aim was to characterize the training, experience, confidence, and perspectives of NRP Providers and Instructors regarding LM use during neonatal resuscitation at birth., Methods: A voluntary anonymous survey was emailed to all NRP Providers and Instructors. Survey items addressed training, experience, confidence, and barriers for LM use during resuscitation. Associations between respondent characteristics and outcomes of both LM experience and confidence were assessed using logistic regression., Results: Between 11/7/22-12/12/22, there were 5,809 survey respondents: 68% were NRP Providers, 55% were nurses, and 87% worked in a hospital setting. Of these, 12% had ever placed a LM during newborn resuscitation, and 25% felt very or completely confident using a LM. In logistic regression, clinical or simulated hands-on training, NRP Instructor role, professional role, and practice setting were all associated with both LM experience and confidence.The three most frequently identified barriers to LM use were insufficient experience (46%), preference for other interfaces (25%), and failure to consider the LM during resuscitation (21%). One-third (33%) reported that LMs are not available where they resuscitate newborns., Conclusion: Few NRP providers and instructors use the LM during neonatal resuscitation. Strategies to increase LM use include hands-on clinical training, outreach promoting the advantages of the LM compared to other interfaces, and improving availability of the LM in delivery settings., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

A statewide landscape analysis was initiated to identify workforce development and educational needs concerning the support of persons with Alzheimer's Disease and Related Dementias (ADRD). Educational programs preparing healthcare professionals were targeted since people with ADRD, and their families/caregivers, often have frequent, ongoing contact with healthcare providers. A literature review and thematic analysis discovered a dearth of research and a lack of consistent competency identification for healthcare education. A crosswalk comparison of various competency models led to the development of a five factor model. A survey based on this model was sent to educators statewide evaluating confidence in ADRD-specific competency attainment in graduates. Descriptive statistics and factor analysis led to a revision of the original five factor model to a three factor model, including competencies in Global Dementia knowledge, Communication, and Safety, each with various sub-competencies. Identifying ADRD-specific competencies for graduating healthcare students is essential. This three factor competency framework will support educational programs in examining curricular offerings and increasing awareness concerning the needs of the ADRD population. Furthermore, using a robust competency model for healthcare education can assist in preparing graduates to address the needs of those with ADRD as well as the needs of the family/caregiving system and environment.

Objective: The devastation of pharmaceutical production facilities from Hurricane Maria caused a national shortage of parenteral amino acids in October 2017. Our institution decreased trophamine in very low birth weight (VLBW) infants and initiated human milk fortification at a lower feeding volume to increase enteral protein intake more quickly. The objective of this study was to assess how protein management during the shortage period affected the incidence of malnutrition., Methods: This was a retrospective cohort study of infants admitted to 2 neonatal intensive care units from June 1, 2017 to May 31, 2018. Infants between 23 and 32 weeks' gestation were included in this study. The primary outcome was the incidence of malnutrition at 14 days, defined as a z score decline of ≥0.8 SDs, in the pre-shortage period compared with the shortage period. Clinical data regarding adverse effects associated with early fortification and pharmacy costs were recorded., Results: There were 68 infants prior to and 65 during the shortage who met inclusion criteria. There was no difference in malnutrition between the pre-shortage and shortage groups; however, a significant increase in malnutrition was observed in infants who did not receive early fortification during the shortage. No difference in time to full enteral feeds or necrotizing enterocolitis was observed with early fortification., Conclusions: Early fortification in VLBW infants receiving less trophamine during the shortage was not associated with an increase in malnutrition. Restricting trophamine in neonates during the shortage allowed for distribution to other critically ill patients., Competing Interests: Disclosures. The authors declare no conflicts or financial interest in any product or service mentioned in the manuscript, including grants, equipment, medications, employment, gifts, and honoraria., (Copyright. Pediatric Pharmacy Association. All rights reserved. For permissions, email: mhelms@pediatricpharmacy.org 2022.)

Objectives: To reduce transfers to the neonatal intensive care unit (NICU) for neonates with opioid withdrawal while also reducing length of stay and pharmacologic intervention, and maintaining standards of safety., Patients and Methods: This was a single-center quality-improvement (QI) initiative in a free-standing maternity hospital comparing outcomes for neonatal opioid withdrawal syndrome (NOWS) before and after a series of QI bundles in infants >36 weeks' gestation age (GA). We compared outcomes to our preintervention period (January, 2013 to December, 2013; n = 42) with outcomes postintervention cycle 1 (October, 2016 to September, 2017; n = 126), and postintervention cycle 2 (November, 2017 to October, 2018; n = 160). Cycle 1 included organizing a multidisciplinary task force who focused on emphasis on nonpharmacologic and dyad-centered care, and also standardized pharmacologic management. Cycle 2 reflects the transition to a functional assessment tool and as-needed morphine administration on the postpartum floor., Results: Transfer to the NICU for management of NOWS dropped from 71.4% before the quality improvement project down to 5.6% (P < 0.001), with the remainder managed on the mother-baby unit. Length of stay decreased from 17.8 days to 7.2 days, and opioid replacement dropped from 60% down to 16% (P < 0.001 for both). There were no adverse events from morphine administration for any of the infants in this series., Conclusions: Our study demonstrates how care can be safely provided to most infants with neonatal opioid withdrawal on a postpartum unit without needing transfer to another unit or a higher level of care facility.

Introduction: Timing of cord clamping and other cord management strategies may improve outcomes at preterm birth. However, it is unclear whether benefits apply to all preterm subgroups. Previous and current trials compare various policies, including time-based or physiology-based deferred cord clamping, and cord milking. Individual participant data (IPD) enable exploration of different strategies within subgroups. Network meta-analysis (NMA) enables comparison and ranking of all available interventions using a combination of direct and indirect comparisons., Objectives: (1) To evaluate the effectiveness of cord management strategies for preterm infants on neonatal mortality and morbidity overall and for different participant characteristics using IPD meta-analysis. (2) To evaluate and rank the effect of different cord management strategies for preterm births on mortality and other key outcomes using NMA., Methods and Analysis: Systematic searches of Medline, Embase, clinical trial registries, and other sources for all ongoing and completed randomised controlled trials comparing cord management strategies at preterm birth (before 37 weeks' gestation) have been completed up to 13 February 2019, but will be updated regularly to include additional trials. IPD will be sought for all trials; aggregate summary data will be included where IPD are unavailable. First, deferred clamping and cord milking will be compared with immediate clamping in pairwise IPD meta-analyses. The primary outcome will be death prior to hospital discharge. Effect differences will be explored for prespecified participant subgroups. Second, all identified cord management strategies will be compared and ranked in an IPD NMA for the primary outcome and the key secondary outcomes. Treatment effect differences by participant characteristics will be identified. Inconsistency and heterogeneity will be explored., Ethics and Dissemination: Ethics approval for this project has been granted by the University of Sydney Human Research Ethics Committee (2018/886). Results will be relevant to clinicians, guideline developers and policy-makers, and will be disseminated via publications, presentations and media releases., Registration Number: Australian New Zealand Clinical Trials Registry (ANZCTR) (ACTRN12619001305112) and International Prospective Register of Systematic Reviews (PROSPERO, CRD42019136640)., Competing Interests: Competing interests: LD, ACK, CDPM, ED, HR, JK, JM, JJ, KF, OA, SH, VS, VD, WE-N and WT-M are Chief Investigators for potentially eligible trials., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY. Published by BMJ.)

Bioinformatic analysis of the putative nuclease domain of the single polypeptide restriction-modification enzyme LlaGI reveals amino acid motifs characteristic of the Escherichia coli methylated DNA-specific Mrr endonuclease. Using mutagenesis, we examined the role of the conserved residues in both DNA translocation and cleavage. Mutations in those residues predicted to play a role in DNA hydrolysis produced enzymes that could translocate on DNA but were either unable to cleave the polynucleotide track or had reduced nuclease activity. Cleavage by LlaGI is not targeted to methylated DNA, suggesting that the conserved motifs in the Mrr domain are a conventional sub-family of the PD-(D/E)XK superfamily of DNA nucleases.

LlaGI is a single polypeptide restriction-modification enzyme encoded on the naturally-occurring plasmid pEW104 isolated from Lactococcus lactis ssp. cremoris W10. Bioinformatics analysis suggests that the enzyme contains domains characteristic of an mrr endonuclease, a superfamily 2 DNA helicase and a gamma-family adenine methyltransferase. LlaGI was expressed and purified from a recombinant clone and its properties characterised. An asymmetric recognition sequence was identified, 5'-CTnGAyG-3' (where n is A, G, C or T and y is C or T). Methylation of the recognition site occurred on only one strand (the non-degenerate dA residue of 5'-CrTCnAG-3' being methylated at the N6 position). Double strand DNA breaks at distant, random sites were only observed when two head-to-head oriented, unmethylated copies of the site were present; single sites or pairs in tail-to-tail or head-to-tail repeat only supported a DNA nicking activity. dsDNA nuclease activity was dependent upon the presence of ATP or dATP. Our results are consistent with a directional long-range communication mechanism that is necessitated by the partial site methylation. In the accompanying manuscript [Smith et al. (2009) The single polypeptide restriction-modification enzyme LlaGI is a self-contained molecular motor that translocates DNA loops], we demonstrate that this communication is via 1-dimensional DNA loop translocation. On the basis of this data and that in the third accompanying manuscript [Smith et al. (2009) An Mrr-family nuclease motif in the single polypeptide restriction-modification enzyme LlaGI], we propose that LlaGI is the prototype of a new sub-classification of Restriction-Modification enzymes, named Type I SP (for Single Polypeptide).

To cleave DNA, the single polypeptide restriction-modification enzyme LlaGI must communicate between a pair of indirectly repeated recognition sites. We demonstrate that this communication occurs by a 1-dimensional route, namely unidirectional dsDNA loop translocation rightward of the specific recognition sequence 5'-CTnGAyG-3' as written (where n is either A, G, C or T and y is either C or T). Motion across thousands of base pairs is catalysed by the helicase domain and requires the hydrolysis of 1.5-2 ATP per base pair. DNA loop extrusion is accompanied by changes in DNA twist consistent with the motor following the helical pitch of the polynucleotide track. LlaGI is therefore an example of a polypeptide that is a completely self-contained, multi-functional molecular machine.

Lactococcus lactis phage P335 is a virulent type phage for the species that bears its name and belongs to the Siphoviridae family. Morphologically, P335 resembled the L. lactis phages TP901-1 and Tuc2009, except for a shorter tail and a different collar/whisker structure. Its 33,613-bp double-stranded DNA genome had 50 open reading frames. Putative functions were assigned to 29 of them. Unlike other sequenced genomes from lactococcal phages belonging to this species, P335 did not have a lysogeny module. However, it did carry a dUTPase gene, the most conserved gene among this phage species. Comparative genomic analyses revealed a high level of identity between the morphogenesis modules of the phages P335, ul36, TP901-1, and Tuc2009 and two putative prophages of L. lactis SK11. Differences were noted in genes coding for receptor-binding proteins, in agreement with their distinct host ranges. Sixteen structural proteins of phage P335 were identified by liquid chromatography-tandem mass spectrometry. A 2.8-kb insertion was recognized between the putative genes coding for the activator of late transcription (Alt) and the small terminase subunit (TerS). Four genes within this region were autonomously late transcribed and possibly under the control of Alt. Three of the four deduced proteins had similarities with proteins from Streptococcus pyogenes prophages, suggesting that P335 acquired this module from another phage genome. The genetic diversity of the P335 species indicates that they are exceptional models for studying the modular theory of phage evolution.

Aim: To compare pH and conductivity used in the determination of growth in reconstituted skim milk (RSM), to determine whether the presence of one or two plasmids in Lactococcus lactis had any influence on growth, and whether AbiS improved bacteriophages resistance of L. lactis., Methods and Results: Conductivity and pH were used to determine growth in RSM. A small increase in the generation time was found with increasing number of plasmids, while their size was unimportant. The introduction of a plasmid-encoding AbiS did only enhance the level of phage resistance significant when other plasmids encoding either AbiS1 or the restriction modification system LlaBIII was present., Conclusions: The earliest detection of growth was observed by measuring pH, rather than conductance. The plasmid-encoded AbiS system has a potential to be used as a phage resistance mechanisms in L. lactis during milk fermentations, especially when combined with other anti-phage mechanisms., Significance and Impact of the Study: This study widened the knowledge about the influence of plasmid introduction on the growth rate of L. lactis, which is important for the construction of new strains. The level of protection against 936 groups of phages was only significant when the mechanism was present together with the RM system LlaBIII.

Various strains of Escherichia coli, isolated from different patients, were screened for type II restriction endonuclease activity. In 1 out of 23 patients, a type II restriction endonuclease activity was found. The restriction endonuclease designated Eco1524I was purified to near homogeneity, based on hydroxyapatite and heparin sepharose chromatography. Eco1524I exhibited endonuclease restriction activity in the pH range from 6.0 to 10.0 (maximum level at pH 8.0) and required Mg2+ as divalent cation. The enzyme was stable till temperature 55 degrees C and pH range from 6.0 to 10.0. Eco1524I recognized the sequence 6-bp palindromic 5'AGG downward arrow CCT 3', producing blunt end and is found to be an isoschizomer of Stu I.

The aim of this work was to identify genes responsible for host recognition in the lactococcal phages sk1 and bIL170 belonging to species 936. These phages have a high level of DNA identity but different host ranges. Bioinformatic analysis indicated that homologous genes, orf18 in sk1 and orf20 in bIL170, could be the receptor-binding protein (RBP) genes, since the resulting proteins were unrelated in the C-terminal part and showed homology to different groups of proteins hypothetically involved in host recognition. Consequently, chimeric bIL170 phages carrying orf18 from sk1 were generated. The recombinant phages were able to form plaques on the sk1 host Lactococcus lactis MG1614, and recombination was verified by PCR analysis directly with the plaques. A polyclonal antiserum raised against the C-terminal part of phage sk1 ORF18 was used in immunogold electron microscopy to demonstrate that ORF18 is located at the tip of the tail. Sequence analysis of corresponding proteins from other lactococcal phages belonging to species 936 showed that the N-terminal parts of the RBPs were very similar, while the C-terminal parts varied, suggesting that the C-terminal part plays a role in receptor binding. The phages investigated could be grouped into sk1-like phages (p2, fd13, jj50, and phi 7) and bIL170-like phages (P008, P113G, P272, and bIL66) on the basis of the homology of their RBPs to the C-terminal part of ORF18 in sk1 and ORF20 in bIL170, respectively. Interestingly, sk1-like phages bind to and infect a defined group of L. lactis subsp. cremoris strains, while bIL170-like phages bind to and infect a defined group of L. lactis subsp. lactis strains.

The aim of this work was to identify genes in Lactococcus lactis subsp. lactis IL1403 and Lactococcus lactis subsp. cremoris Wg2 important for adsorption of the 936-species phages bIL170 and phi 645, respectively. Random insertional mutagenesis of the two L. lactis strains was carried out with the vector pGh9:ISS1, and integrants that were resistant to phage infection and showed reduced phage adsorption were selected. In L. lactis IL1403 integration was obtained in the ycaG and rgpE genes, whereas in L. lactis Wg2 integration was obtained in two genes homologous to ycbC and ycbB of L. lactis IL1403. rgpE and ycbB encode putative glycosyltransferases, whereas ycaG and ycbC encode putative membrane-spanning proteins with unknown functions. Interestingly, ycaG, rgpE, ycbC, and ycbB are all part of the same operon in L. lactis IL1403. This operon is probably involved in biosynthesis and transport of cell wall polysaccharides (WPS). Binding and infection studies showed that phi645 binds to and infects L. lactis Wg2, L. lactis IL1403, and L. lactis IL1403 strains with pGh9:ISS1 integration in ycaG and rgpE, whereas bIL170 binds to and infects only L. lactis IL1403 and cannot infect Wg2. These results indicate that phi 645 binds to a WPS structure present in both L. lactis IL1403 and L. lactis Wg2, whereas bIL170 binds to another WPS structure not present in L. lactis Wg2. Binding of bIL170 and phi 645 to different WPS structures was supported by alignment of the receptor-binding proteins of bIL170 and phi 645 that showed no homology in the C-terminal part.

The type II restriction-modification (R-M) system LlaDII isolated from Lactococcus lactis contains two tandemly arranged genes, llaDIIR and llaDIIM, encoding a restriction endonuclease (REase) and a methyltransferase (MTase), respectively. Interestingly, two LlaDII recognition sites are present in the llaDIIM promoter region, suggesting that they may influence the activity of the promoter through methylation status. In this study, separate promoters for llaDIIR and llaDIIM were identified, and the regulation of the two genes at the transcriptional level was investigated. DNA fragments containing the putative promoters were cloned in a promoter probe vector and tested for activity in the presence and absence of the active MTase. The level of expression of the MTase was 5- to 10-fold higher than the level of expression of the REase. The results also showed that the presence of M.LlaDII reduced the in vivo expression of the llaDIIM promoter (P(llaDIIM)) up to 1,000-fold, whereas the activity of the llaDIIR promoter (P(llaDIIR)) was not affected. Based on site-specific mutations it was shown that both of the LlaDII recognition sites within P(llaDIIM) are required to obtain complete repression of transcriptional activity. No regulation was found for llaDIIR, which appears to be constitutively expressed.

A nomenclature is described for restriction endonucleases, DNA methyltransferases, homing endonucleases and related genes and gene products. It provides explicit categories for the many different Type II enzymes now identified and provides a system for naming the putative genes found by sequence analysis of microbial genomes.

LlaBIII, isolated from the naturally occurring plasmid pJW566 from Lactococcus lactis subsp. cremoris W56, encoded a restriction and modification (R/M) system. The plasmid pJK1 carrying the R/M system LlaBIII was transformed into L. lactis IL1403 with type I R/M system located on chromosome and the strain MG1614[pAW601] with AbiS gene on plasmid pAW601, respectively. The transformants obtained showed stacking resistance against bacteriophages. The plasmid pJK1 was transformed into industrial strains L. lactis SMQ86 and CHCC2281, the transformants showed the EOP of the bacteriophages decreased by 10(-3) and 10(-5), respectively. The results indicated that the R/M system LlaBIII could protect strains from bacteriophages in dairy fermentation.

The naturally occurring 12.1-kb plasmid, pEW104, in Lactococcus lactis ssp. cremoris W10 was found to confer decreased bacteriophage sensitivity to its host. Plasmid pEW104 encodes a non-classic restriction and modification (R/M) system, named LlaGI, consisting of only one single polypeptide. Analysis of the amino acid sequence revealed the presence of a catalytic motif and seven helicase-like motifs (DEAD-box motifs) characteristic of type I and III endonucleases, followed by four conserved methylase motifs characteristic of adenine-methylases. A comparison between LlaGI and the very similar R/M system, LlaBIII, suggests that the C-terminal region of LlaGI, apparently containing no known motifs, could possibly specify target DNA recognition. Conceivably, the LlaGI gene is included in the operon of the plasmid replication machinery. Finally, it is proposed that LlaGI represents a variant of the type I R/M systems.

A novel type I restriction-modification specificity subunit, S. LlaW12I, has been identified on the naturally occurring 8.0-kb plasmid pAW122 in the lactic acid bacterium Lactococcus lactis subsp. cremoris W12. Presence of the HsdS protein together with a complete type I restriction-modification system conferred increased phage restriction to the host, indicating exchange of specificity subunits. Sequence analysis showed that the S.LlaW12I subunit is most probably of type IC. Presumably, the hsdS gene is organized together with the repB gene on one transcriptional unit., (Copyright 2000 Academic Press.)

The temperate phage TPW22, induced from Lactococcus lactis subsp. cremoris W22, and the evolutionarily interesting integrase of this phage were characterized. Phage TPW22 was propagated lytically on L. lactis subsp. cremoris 3107, which could also be lysogenized by site-specific integration. The attachment site (attP), 5'-TAAGGCGACGGTCG-3', of phage TPW22 was present on a 7.5-kb EcoRI fragment, a 3.4-kb EcoRI-HindIII fragment of which was sequenced. Sequence information revealed the presence of an integrase gene (int). The deduced amino acid sequence showed 42 and 28% identity with integrases of streptococcal and lactococcal phages, respectively. The identities with these integrase-encoding genes were 52 and 45%, respectively, at the nucleotide level. This could indicate horizontal gene transfer. A stable integration vector containing attP and int was constructed, and integration in L. lactis subsp. cremoris MG1363 was obtained. The existence of an exchangeable lactococcal phage integration module was suggested. The proposed module covers the phage attachment site, the integrase gene, and surrounding factor-independent terminator structures. The phages phiLC3, TP901-1, and TPW22 all have different versions of this module. Phylogenetically, the TPW22 Int links the phiLC3 lactococcal integrase with known Streptococcus thermophilus integrases.

The LlaDII restriction/modification (R/M) system was found on the naturally occurring 8.9-kb plasmid pHW393 in Lactococcus lactis subsp. cremoris W39. A 2.4-kb PstI-EcoRI fragment inserted into the Escherichia coli-L. lactis shuttle vector pCI3340 conferred to L. lactis LM2301 and L. lactis SMQ86 resistance against representatives of the three most common lactococcal phage species: 936, P335, and c2. The LlaDII endonuclease was partially purified and found to recognize and cleave the sequence 5'-GC decreases NGC-3', where the arrow indicates the cleavage site. It is thus an isoschizomer of the commercially available restriction endonuclease Fnu4HI. Sequencing of the 2.4-kb PstI-EcoRI fragment revealed two open reading frames arranged tandemly and separated by a 105-bp intergenic region. The endonuclease gene of 543 bp preceded the methylase gene of 954 bp. The deduced amino acid sequence of the LlaDII R/M system showed high homology to that of its only sequenced isoschizomer, Bsp6I from Bacillus sp. strain RFL6, with 41% identity between the endonucleases and 60% identity between the methylases. The genetic organizations of the LlaDII and Bsp6I R/M systems are identical. Both methylases have two recognition sites (5'-GCGGC-3' and 5'-GCCGC-3') forming a putative stemloop structure spanning part of the presumed -35 sequence and part of the intervening region between the -35 and -10 sequences. Alignment of the LlaDII and Bsp6I methylases with other m5C methylases showed that the protein primary structures possessed the same organization.