Control of a gene transfer agent cluster in Caulobacter crescentus by transcriptional activation and anti-termination.

Gene Transfer Agents (GTAs) are phage-like particles that cannot self-multiply and be infectious. Caulobacter crescentus, a bacterium best known as a model organism to study bacterial cell biology and cell cycle regulation, has recently been demonstrated to produce bona fide GTA particles (CcGTA). Since C. crescentus ultimately die to release GTA particles, the production of GTA particles must be tightly regulated and integrated with the host physiology to prevent a collapse in cell population. Two direct activators of the CcGTA biosynthetic gene cluster, GafY and GafZ, have been identified, however, it is unknown how GafYZ controls transcription or how they coordinate gene expression of the CcGTA gene cluster with other accessory genes elsewhere on the genome for complete CcGTA production. Here, we show that the CcGTA gene cluster is transcriptionally co-activated by GafY, integration host factor (IHF), and by GafZ-mediated transcription anti-termination. We present evidence that GafZ is a transcription anti-terminator that likely forms an anti-termination complex with RNA polymerase, NusA, NusG, and NusE to bypass transcription terminators within the 14 kb CcGTA cluster. Overall, we reveal a two-tier regulation that coordinates the synthesis of GTA particles in C. crescentus.

A new species of Habrophorula from Vietnam and an updated key to species of the genus (Hymenoptera, Apidae).

The rare bee genus Habrophorula Lieftinck, 1974 is recorded for the first time from Vietnam. The genus is represented by a new species, Habrophorulabelladeceptrix Tran, Engel & Nguyen, sp. nov., from Cao Bang Province and can be most easily confused with H.nigripes Wu from China. The species is most easily differentiated by the unique form of the male terminalia but can also be distinguished by differences in integumental and setal coloration. A revised key is provided to the species of the genus. Females of the new species were collected at flowers of Saurauiaroxburghii Wall. and Saurauianapaulensis DC. (Actinidiaceae); males were collected at flowers of Lantanacamara L. (Verbenaceae).

Widespread prevalence of a methylation-dependent switch to activate an essential DNA damage response in bacteria.

DNA methylation plays central roles in diverse cellular processes, ranging from error-correction during replication to regulation of bacterial defense mechanisms. Nevertheless, certain aberrant methylation modifications can have lethal consequences. The mechanisms by which bacteria detect and respond to such damage remain incompletely understood. Here, we discover a highly conserved but previously uncharacterized transcription factor (Cada2), which orchestrates a methylation-dependent adaptive response in Caulobacter. This response operates independently of the SOS response, governs the expression of genes crucial for direct repair, and is essential for surviving methylation-induced damage. Our molecular investigation of Cada2 reveals a cysteine methylation-dependent posttranslational modification (PTM) and mode of action distinct from its Escherichia coli counterpart, a trait conserved across all bacteria harboring a Cada2-like homolog instead. Extending across the bacterial kingdom, our findings support the notion of divergence and coevolution of adaptive response transcription factors and their corresponding sequence-specific DNA motifs. Despite this diversity, the ubiquitous prevalence of adaptive response regulators underscores the significance of a transcriptional switch, mediated by methylation PTM, in driving a specific and essential bacterial DNA damage response.

The potter wasp genus Allorhynchium from Vietnam, with descriptions of three new species and a new country record (Hymenoptera, Vespidae, Eumeninae).

Species of the potter wasp genus Allorhynchium van der Vecht (Eumeninae: Odynerini) occurring in Vietnam are presented. Seven species have been recorded from Vietnam. Of them, three species are described as new to science: Allorhynchiumlatum Nguyen, Tran & MT Nguyen, sp. nov., A.moerum Nguyen & AD Nguyen, sp. nov., and A.setosum Nguyen & Engel, sp. nov., and one species, A.argentatum (Fabricius, 1804), is recorded from Vietnam for the first time. An updated key to the Oriental species of the genus is presented.

The bee genus Anthidiellum in Vietnam: descriptions of five new species and the first male of Anthidiellumcoronum (Hymenoptera, Megachilidae).

The Vietnamese fauna of bees in the Anthidiellum Cockerell (Megachilinae, Anthidiini) is reviewed. Seven species are recognized, representing two subgenera. Five new species are described and figured as: Anthidiellum (Clypanthidium) nahang Tran, Engel & Nguyen, sp. nov., A. (Pycnanthidium) ayun Tran, Engel & Nguyen, sp. nov., A. (P.) chumomray Tran, Engel & Nguyen, sp. nov., A. (P.) flavaxilla Tran, Engel & Nguyen, sp. nov., and A. (P.) cornu Tran, Engel & Nguyen, sp. nov. from the northern and central highlands of Vietnam. Two previously described species are newly recorded for the fauna: A. (P.) carinatum (Wu) and A. (P.) coronum (Wu), with the male of the latter species described and illustrated for the first time. An identification key is provided for all species of Anthidiellum occurring in Vietnam.

Prophage-like gene transfer agents promote Caulobacter crescentus survival and DNA repair during stationary phase.

Gene transfer agents (GTAs) are prophage-like entities found in many bacterial genomes that cannot propagate themselves and instead package approximately 5 to 15 kbp fragments of the host genome that can then be transferred to related recipient cells. Although suggested to facilitate horizontal gene transfer (HGT) in the wild, no clear physiological role for GTAs has been elucidated. Here, we demonstrate that the α-proteobacterium Caulobacter crescentus produces bona fide GTAs. The production of Caulobacter GTAs is tightly regulated by a newly identified transcription factor, RogA, that represses gafYZ, the direct activators of GTA synthesis. Cells lacking rogA or expressing gafYZ produce GTAs harboring approximately 8.3 kbp fragment of the genome that can, after cell lysis, be transferred into recipient cells. Notably, we find that GTAs promote the survival of Caulobacter in stationary phase and following DNA damage by providing recipient cells a template for homologous recombination-based repair. This function may be broadly conserved in other GTA-producing organisms and explain the prevalence of this unusual HGT mechanism.

A new genus of minute stingless bees from Southeast Asia (Hymenoptera, Apidae).

A new genus of minute stingless bees (Meliponini: Hypotrigonina) is described from Southeast Asia. Ebaiotrigona Engel & Nguyen, gen. nov., is based on the type species Lisotrigonacarpenteri Engel, recorded from Vietnam, Thailand, Laos, Cambodia, and southern China. The species was previously considered an enigmatic member of Lisotrigona Moure, but is removed to a new genus based on its unique male terminalia that differs considerably from that of Lisotrigona and instead shares resemblances with Austroplebeia Moure, and the presence of yellow maculation (also similar to that of Austroplebeia). It is possible that Ebaiotrigona is the extant sister group of Austroplebeia, but this requires confirmation by future phylogenetic analyses. Based on available biological observations, Ebaiotrigonacarpenteri could not be confirmed as lachryphagous as is well documented from the tear-drinking species of Lisotrigona and Pariotrigona Moure.

First occurrence of the little-known genus Noteriades (Hymenoptera, Megachilidae) from Vietnam: discovery of a new species and a key to the Southeast Asian fauna.

The little-known megachiline genus Noteriades Cockerell, 1931 is recorded from Vietnam for the first time. A new species, Noteriadeshangkia Tran, Engel & Nguyen sp. nov. is described and figured based on a series of females collected from the provinces of the northern and central highlands of Vietnam. The genus is briefly discussed and a new subtribe is established, Noteriadina Engel, Tran & Nguyen subtrib. nov. of Megachilini. Lastly, an identification key and distribution map are provided for those species occurring in Southeast Asia.

Two new species of Trichrysis Lichtenstein (Hymenoptera: Chrysididae) from Vietnam and China, with taxonomic notes on other species from the Oriental region.

Two new species of the genus Trichrysis Lichtenstein, belonging to the cyanea species group, are described from northeastern Vietnam (Cao Bang Province) and China (Yunnan Province) as new for science: T. sinica Rosa, Nguyen & Wisniowski, sp. nov., and T. striata Nguyen, Wisniowski & Rosa, sp. nov. The redescription of T. tonkinensis (Mocsáry, 1914) and additional descriptive characters for T. chamchuensis Wisniowski & Nguyen, 2020 are provided.

A CTP-dependent gating mechanism enables ParB spreading on DNA.

Proper chromosome segregation is essential in all living organisms. The ParA-ParB-parS system is widely employed for chromosome segregation in bacteria. Previously, we showed that Caulobacter crescentus ParB requires cytidine triphosphate to escape the nucleation site parS and spread by sliding to the neighboring DNA (Jalal et al., 2020). Here, we provide the structural basis for this transition from nucleation to spreading by solving co-crystal structures of a C-terminal domain truncated C. crescentus ParB with parS and with a CTP analog. Nucleating ParB is an open clamp, in which parS is captured at the DNA-binding domain (the DNA-gate). Upon binding CTP, the N-terminal domain (NTD) self-dimerizes to close the NTD-gate of the clamp. The DNA-gate also closes, thus driving parS into a compartment between the DNA-gate and the C-terminal domain. CTP hydrolysis and/or the release of hydrolytic products are likely associated with reopening of the gates to release DNA and recycle ParB. Overall, we suggest a CTP-operated gating mechanism that regulates ParB nucleation, spreading, and recycling.

CTP regulates membrane-binding activity of the nucleoid occlusion protein Noc.

ATP- and GTP-dependent molecular switches are extensively used to control functions of proteins in a wide range of biological processes. However, CTP switches are rarely reported. Here, we report that a nucleoid occlusion protein Noc is a CTPase enzyme whose membrane-binding activity is directly regulated by a CTP switch. In Bacillus subtilis, Noc nucleates on 16 bp NBS sites before associating with neighboring non-specific DNA to form large membrane-associated nucleoprotein complexes to physically occlude assembly of the cell division machinery. By in vitro reconstitution, we show that (1) CTP is required for Noc to form the NBS-dependent nucleoprotein complex, and (2) CTP binding, but not hydrolysis, switches Noc to a membrane-active state. Overall, we suggest that CTP couples membrane-binding activity of Noc to nucleoprotein complex formation to ensure productive recruitment of DNA to the bacterial cell membrane for nucleoid occlusion activity.

Contribution to the genus Pararrhynchium de Saussure (Hymenoptera: Vespidae: Eumeninae), with description of two new species from Vietnam.

Two new species of the genus Pararrhynchium de Saussure are described as new to science: P. simsanum Nguyen and Tran, sp. nov. from Bat Xat, Lao Cai province, and P. sparsum Nguyen and Ljubomirov, sp. nov. from Ham Yen, Tuyen Quang province. An updated key to all known species of the genus is also presented.

Diversification of DNA-Binding Specificity by Permissive and Specificity-Switching Mutations in the ParB/Noc Protein Family.

Specific interactions between proteins and DNA are essential to many biological processes. Yet, it remains unclear how the diversification in DNA-binding specificity was brought about, and the mutational paths that led to changes in specificity are unknown. Using a pair of evolutionarily related DNA-binding proteins, each with a different DNA preference (ParB [Partitioning Protein B] and Noc [Nucleoid Occlusion Factor], which both play roles in bacterial chromosome maintenance), we show that specificity is encoded by a set of four residues at the protein-DNA interface. Combining X-ray crystallography and deep mutational scanning of the interface, we suggest that permissive mutations must be introduced before specificity-switching mutations to reprogram specificity and that mutational paths to new specificity do not necessarily involve dual-specificity intermediates. Overall, our results provide insight into the possible evolutionary history of ParB and Noc and, in a broader context, might be useful for understanding the evolution of other classes of DNA-binding proteins.

Additional knowledge respecting taxonomy of the social wasp genus Ropalidia (Hymenoptera: Vespidae: Polistinae) from Vietnam, with new records of three species and an updated key to species.

Species composition of social wasps of the genus Ropalidia from Vietnam is reported, with 24 species having been recorded. Of those, three species are newly recorded for the Vietnamese fauna, namely Ropalidia binghami van der Vecht, 1941, R. parartifex Tan van Achterberg, 2014 and R. variegata (Smith, 1852). The presence of R. sumatrae (Weber, 1801) in Vietnam is confirmed. An updated key to all recorded species of the genus Ropalidia from Vietnam is also provided.

ParB spreading on DNA requires cytidine triphosphate in vitro.

In all living organisms, it is essential to transmit genetic information faithfully to the next generation. The SMC-ParAB-parS system is widely employed for chromosome segregation in bacteria. A DNA-binding protein ParB nucleates on parS sites and must associate with neighboring DNA, a process known as spreading, to enable efficient chromosome segregation. Despite its importance, how the initial few ParB molecules nucleating at parS sites recruit hundreds of further ParB to spread is not fully understood. Here, we reconstitute a parS-dependent ParB spreading event using purified proteins from Caulobacter crescentus and show that CTP is required for spreading. We further show that ParB spreading requires a closed DNA substrate, and a DNA-binding transcriptional regulator can act as a roadblock to attenuate spreading unidirectionally in vitro. Our biochemical reconstitutions recapitulate many observed in vivo properties of ParB and opens up avenues to investigate the interactions between ParB-parS with ParA and SMC.

Defining the regulon of genes controlled by σE , a key regulator of the cell envelope stress response in Streptomyces coelicolor.

The extracytoplasmic function (ECF) σ factor, σE , is a key regulator of the cell envelope stress response in Streptomyces coelicolor. Although its role in maintaining cell wall integrity has been known for over a decade, a comprehensive analysis of the genes under its control has not been undertaken. Here, using a combination of chromatin immunoprecipitation-sequencing (ChIP-seq), microarray transcriptional profiling and bioinformatic analysis, we attempt to define the σE regulon. Approximately half of the genes identified encode proteins implicated in cell envelope function. Seventeen novel targets were validated by S1 nuclease mapping or in vitro transcription, establishing a σE -binding consensus. Subsequently, we used bioinformatic analysis to look for conservation of the σE target promoters identified in S. coelicolor across 19 Streptomyces species. Key proteins under σE control across the genus include the actin homolog MreB, three penicillin-binding proteins, two L,D-transpeptidases, a LytR-CpsA-Psr-family protein predicted to be involved in cell wall teichoic acid deposition and a predicted MprF protein, which adds lysyl groups to phosphatidylglycerol to neutralize membrane surface charge. Taken together, these analyses provide biological insight into the σE -mediated cell envelope stress response in the genus Streptomyces.

The MerR-like protein BldC binds DNA direct repeats as cooperative multimers to regulate Streptomyces development.

Streptomycetes are notable for their complex life cycle and production of most clinically important antibiotics. A key factor that controls entry into development and the onset of antibiotic production is the 68-residue protein, BldC. BldC is a putative DNA-binding protein related to MerR regulators, but lacks coiled-coil dimerization and effector-binding domains characteristic of classical MerR proteins. Hence, the molecular function of the protein has been unclear. Here we show that BldC is indeed a DNA-binding protein and controls a regulon that includes other key developmental regulators. Intriguingly, BldC DNA-binding sites vary significantly in length. Our BldC-DNA structures explain this DNA-binding capability by revealing that BldC utilizes a DNA-binding mode distinct from MerR and other known regulators, involving asymmetric head-to-tail oligomerization on DNA direct repeats that results in dramatic DNA distortion. Notably, BldC-like proteins radiate throughout eubacteria, establishing BldC as the founding member of a new structural family of regulators.

Permissive zones for the centromere-binding protein ParB on the Caulobacter crescentus chromosome.

Proper chromosome segregation is essential in all living organisms. In Caulobacter crescentus, the ParA-ParB-parS system is required for proper chromosome segregation and cell viability. The bacterial centromere-like parS DNA locus is the first to be segregated following chromosome replication. parS is bound by ParB protein, which in turn interacts with ParA to partition the ParB-parS nucleoprotein complex to each daughter cell. Here, we investigated the genome-wide distribution of ParB on the Caulobacter chromosome using a combination of in vivo chromatin immunoprecipitation (ChIP-seq) and in vitro DNA affinity purification with deep sequencing (IDAP-seq). We confirmed two previously identified parS sites and discovered at least three more sites that cluster ∼8 kb from the origin of replication. We showed that Caulobacter ParB nucleates at parS sites and associates non-specifically with ∼10 kb flanking DNA to form a high-order nucleoprotein complex on the left chromosomal arm. Lastly, using transposon mutagenesis coupled with deep sequencing (Tn-seq), we identified a ∼500 kb region surrounding the native parS cluster that is tolerable to the insertion of a second parS cluster without severely affecting cell viability. Our results demonstrate that the genomic distribution of parS sites is highly restricted and is crucial for chromosome segregation in Caulobacter.

SMC Progressively Aligns Chromosomal Arms in Caulobacter crescentus but Is Antagonized by Convergent Transcription.

The structural maintenance of chromosomes (SMC) complex plays an important role in chromosome organization and segregation in most living organisms. In Caulobacter crescentus, SMC is required to align the left and the right arms of the chromosome that run in parallel down the long axis of the cell. However, the mechanism of SMC-mediated alignment of chromosomal arms remains elusive. Here, using genome-wide methods and microscopy of single cells, we show that Caulobacter SMC is recruited to the centromeric parS site and that SMC-mediated arm alignment depends on the chromosome-partitioning protein ParB. We provide evidence that SMC likely tethers the parS-proximal regions of the chromosomal arms together, promoting arm alignment. Furthermore, we show that highly transcribed genes near parS that are oriented against SMC translocation disrupt arm alignment, suggesting that head-on transcription interferes with SMC translocation. Our results demonstrate a tight interdependence of bacterial chromosome organization and global patterns of transcription.

A Conserved Pattern of Primer-Dependent Transcription Initiation in Escherichia coli and Vibrio cholerae Revealed by 5' RNA-seq.

Transcription initiation that involves the use of a 2- to ~4-nt oligoribonucleotide primer, "primer-dependent initiation," (PDI) has been shown to be widely prevalent at promoters of genes expressed during the stationary phase of growth in Escherichia coli. However, the extent to which PDI impacts E. coli physiology, and the extent to which PDI occurs in other bacteria is not known. Here we establish a physiological role for PDI in E. coli as a regulatory mechanism that modulates biofilm formation. We further demonstrate using high-throughput sequencing of RNA 5' ends (5' RNA-seq) that PDI occurs in the pathogenic bacterium Vibrio cholerae. A comparative global analysis of PDI in V. cholerae and E. coli reveals that the pattern of PDI is strikingly similar in the two organisms. In particular, PDI is detected in stationary phase, is not detected in exponential phase, and is preferentially apparent at promoters carrying the sequence T-1A+1 or G-1G+1 (where position +1 corresponds to the position of de novo initiation). Our findings demonstrate a physiological role for PDI and suggest PDI may be widespread among Gammaproteobacteria. We propose that PDI in both E. coli and V. cholerae occurs though a growth phase-dependent process that leads to the preferential generation of the linear dinucleotides 5´-UA-3´ and 5´-GG-3´.