Molecular mechanisms and biological consequences of the production of non-canonical D-amino acids in bacteria

Most bacteria possess a vital net-like macromolecule – peptidoglycan (PG). PG encases bacteria around the cytoplasmic membrane to withstand the high internal turgor pressure and thereby protect the cell from bursting. In addition, PG is a major morphological determinant of bacteria being both required and sufficient to maintain cell shape. During cell growth PG hydrolysis and synthesis are tightly controlled to keep proper cell shape and integrity at all times. Given the essentiality of PG for bacterial growth and survival, the synthesis of this polymer is a major target of many natural and synthetic antibiotics (e.g. penicillins, glycopeptides). For a long time, PG composition was considered to be conserved and static, however it’s now being recognized as a dynamic and plastic macromolecule. The structure and chemistry of PG is influenced by a myriad of environmental cues that include interkingdom/interspecies interactions. Recently, it was found that a wide set of non-canonical D-amino acids (D-amino acids different from D-Ala and D-Glu, NCDAAs) are produced and released to the extracellular milieu by diverse bacteria. In Vibrio cholerae these NCDAAs are produced by broad-spectrum racemase enzyme (BsrV) and negatively regulate PG synthesis through their incorporation into PG. We have shown that in addition to D-Met and D-Leu, which were reported previously, V. cholerae also releases high amounts of D-Arg, which inhibits a broader range of phylogenetically diverse bacteria. Thus, NCDAAs affect not only the producer, but might target other species within the same environmental niche. However, in contrast to D-Met, D-Arg targets cell wall independent pathways.  We have shown that non-proteinogenic amino acids also can be racemized by Bsr. A plant amino acid L-canavanine (L-CAN) is converted into D-CAN by a broad-spectrum amino acid racemase (BSAR) of the soil bacterium Pseudomonas putida and subsequently released to the environment. D-CAN gets highly incorporated into the PG of Rhizobiales (such as Agrobacterium tumefaciens, Sinorhizobium meliloti) thereby affecting the overall PG structure, bacterial morphogenesis and growth fitness. We found that detrimental effect of D-CAN in A. tumefaciens can be suppressed by a single amino acid substitution in the cell division PG transpeptidase penicillin-binding protein 3a (PBP3a).  Rhizobiales are a polar-growing species that encode multiple LD-transpeptidases (LDTs), enzymes that normally perform PG crosslinking, but that can also incorporate NCDAAs into termini of the PG peptides. As these species incorporate high amounts of D-CAN in their PG, we hypothesized that LDTs might represent the main path used by NCDAAs to edit A. tumefaciens’ PG and cause their detrimental effects. Therefore, we decided to further explore the significance of LDT proteins for growth and morphogenesis in A. tumefaciens. While in the Gram-negative model organism E. coli LDT proteins are non-essential under standard laboratory conditions, we found that A. tumefaciens needs at least one LDT for growth out of the 14 putative LDTs encoded in its genome. Moreover, clustering the LDT proteins based on their sequence similarity revealed that A. tumefaciens has 7 LDTs that are exclusively present among Rhizobiales. Interestingly, the loss of this group of LDTs (but not the rest) leads to reduced growth, lower PG crosslinkage and rounded cell phenotype, which suggests that this group of Rhizobiales- specific LDTs have a major role in maintaining LD-crosslinking homeostasis, which in turn is important for cell elongation and proper shape maintenance in A. tumefaciens

Architecture of Interaction: Re-thinking Moscow's Apartments

During the social transformation in the 1950’s through to the late 1980’s, thousands of Khrushchyovka* were built in Moscow to cater for the social housing demand after the war. After nearly 70 years of occupation these housing developments and homes have become outdated, overrun and irreparable. The current government strategy is to forcibly relocate families from Khrushchyovka to apartments of ‘equal size’ on the outskirts of Moscow. Even in the newer developments high-density living is seen as temporary and confined to specific life stages. This view is informed by the lack of social space and increase of narrow and dark spaces which are seen to compromise health and safety and is the result of ridged and stringent planning. This thesis proposes to incorporate the principles of social interaction such as passive contact and low intensity interaction opportunities; which is merely the ability to meet, see and hear and thereby to improve living conditions and bring back a sense community to high-density urban housing in Moscow. It argues that a new planning and design framework for high-density housing through low intensity social contact can promote greater social engagement, which can contribute to people’s wellbeing and happiness. The development of an urban and architectural framework for greater social interaction will look at creating opportunities for the interaction of people in public and semi-public space. This will provide a method for understanding how social contact can be combined with the provision of private and public space, while looking to promote safety, comfort and healthier living environments. It also recognises the importance of the public and semi-private spaces being governed by the residents and not private entities or individuals. Khrushchyovka [Хрущёвка] - Housing built in the 1950s-1960s and named after Nikita Khrushchev (First Secretary of the Central Committee of the Communist Party of the Soviet Union) 1953-1964

Molecular mechanisms and biological consequences of the production of non-canonical D-amino acids in bacteria

Most bacteria possess a vital net-like macromolecule – peptidoglycan (PG). PG encases bacteria around the cytoplasmic membrane to withstand the high internal turgor pressure and thereby protect the cell from bursting. In addition, PG is a major morphological determinant of bacteria being both required and sufficient to maintain cell shape. During cell growth PG hydrolysis and synthesis are tightly controlled to keep proper cell shape and integrity at all times. Given the essentiality of PG for bacterial growth and survival, the synthesis of this polymer is a major target of many natural and synthetic antibiotics (e.g. penicillins, glycopeptides). For a long time, PG composition was considered to be conserved and static, however it’s now being recognized as a dynamic and plastic macromolecule. The structure and chemistry of PG is influenced by a myriad of environmental cues that include interkingdom/interspecies interactions. Recently, it was found that a wide set of non-canonical D-amino acids (D-amino acids different from D-Ala and D-Glu, NCDAAs) are produced and released to the extracellular milieu by diverse bacteria. In Vibrio cholerae these NCDAAs are produced by broad-spectrum racemase enzyme (BsrV) and negatively regulate PG synthesis through their incorporation into PG. We have shown that in addition to D-Met and D-Leu, which were reported previously, V. cholerae also releases high amounts of D-Arg, which inhibits a broader range of phylogenetically diverse bacteria. Thus, NCDAAs affect not only the producer, but might target other species within the same environmental niche. However, in contrast to D-Met, D-Arg targets cell wall independent pathways.  We have shown that non-proteinogenic amino acids also can be racemized by Bsr. A plant amino acid L-canavanine (L-CAN) is converted into D-CAN by a broad-spectrum amino acid racemase (BSAR) of the soil bacterium Pseudomonas putida and subsequently released to the environment. D-CAN gets highly incorporated into the PG of Rhizobiales (such as Agrobacterium tumefaciens, Sinorhizobium meliloti) thereby affecting the overall PG structure, bacterial morphogenesis and growth fitness. We found that detrimental effect of D-CAN in A. tumefaciens can be suppressed by a single amino acid substitution in the cell division PG transpeptidase penicillin-binding protein 3a (PBP3a).  Rhizobiales are a polar-growing species that encode multiple LD-transpeptidases (LDTs), enzymes that normally perform PG crosslinking, but that can also incorporate NCDAAs into termini of the PG peptides. As these species incorporate high amounts of D-CAN in their PG, we hypothesized that LDTs might represent the main path used by NCDAAs to edit A. tumefaciens’ PG and cause their detrimental effects. Therefore, we decided to further explore the significance of LDT proteins for growth and morphogenesis in A. tumefaciens. While in the Gram-negative model organism E. coli LDT proteins are non-essential under standard laboratory conditions, we found that A. tumefaciens needs at least one LDT for growth out of the 14 putative LDTs encoded in its genome. Moreover, clustering the LDT proteins based on their sequence similarity revealed that A. tumefaciens has 7 LDTs that are exclusively present among Rhizobiales. Interestingly, the loss of this group of LDTs (but not the rest) leads to reduced growth, lower PG crosslinkage and rounded cell phenotype, which suggests that this group of Rhizobiales- specific LDTs have a major role in maintaining LD-crosslinking homeostasis, which in turn is important for cell elongation and proper shape maintenance in A. tumefaciens

Architecture of Interaction: Re-thinking Moscow's Apartments

During the social transformation in the 1950’s through to the late 1980’s, thousands of Khrushchyovka* were built in Moscow to cater for the social housing demand after the war. After nearly 70 years of occupation these housing developments and homes have become outdated, overrun and irreparable. The current government strategy is to forcibly relocate families from Khrushchyovka to apartments of ‘equal size’ on the outskirts of Moscow. Even in the newer developments high-density living is seen as temporary and confined to specific life stages. This view is informed by the lack of social space and increase of narrow and dark spaces which are seen to compromise health and safety and is the result of ridged and stringent planning. This thesis proposes to incorporate the principles of social interaction such as passive contact and low intensity interaction opportunities; which is merely the ability to meet, see and hear and thereby to improve living conditions and bring back a sense community to high-density urban housing in Moscow. It argues that a new planning and design framework for high-density housing through low intensity social contact can promote greater social engagement, which can contribute to people’s wellbeing and happiness. The development of an urban and architectural framework for greater social interaction will look at creating opportunities for the interaction of people in public and semi-public space. This will provide a method for understanding how social contact can be combined with the provision of private and public space, while looking to promote safety, comfort and healthier living environments. It also recognises the importance of the public and semi-private spaces being governed by the residents and not private entities or individuals. Khrushchyovka [Хрущёвка] - Housing built in the 1950s-1960s and named after Nikita Khrushchev (First Secretary of the Central Committee of the Communist Party of the Soviet Union) 1953-1964

Architecture of Interaction: Re-thinking Moscow's Apartments

During the social transformation in the 1950’s through to the late 1980’s, thousands of Khrushchyovka* were built in Moscow to cater for the social housing demand after the war. After nearly 70 years of occupation these housing developments and homes have become outdated, overrun and irreparable. The current government strategy is to forcibly relocate families from Khrushchyovka to apartments of ‘equal size’ on the outskirts of Moscow. Even in the newer developments high-density living is seen as temporary and confined to specific life stages. This view is informed by the lack of social space and increase of narrow and dark spaces which are seen to compromise health and safety and is the result of ridged and stringent planning. This thesis proposes to incorporate the principles of social interaction such as passive contact and low intensity interaction opportunities; which is merely the ability to meet, see and hear and thereby to improve living conditions and bring back a sense community to high-density urban housing in Moscow. It argues that a new planning and design framework for high-density housing through low intensity social contact can promote greater social engagement, which can contribute to people’s wellbeing and happiness. The development of an urban and architectural framework for greater social interaction will look at creating opportunities for the interaction of people in public and semi-public space. This will provide a method for understanding how social contact can be combined with the provision of private and public space, while looking to promote safety, comfort and healthier living environments. It also recognises the importance of the public and semi-private spaces being governed by the residents and not private entities or individuals. Khrushchyovka [Хрущёвка] - Housing built in the 1950s-1960s and named after Nikita Khrushchev (First Secretary of the Central Committee of the Communist Party of the Soviet Union) 1953-1964.</p

INTERTEXTUAL ELEMENTS IN THE WORKS OF E. AKULIN

Артыкул прысвечаны аналізу інтэртэкстуальных элементаў у паэзіі Эдуарда Акуліна. Навізна працы заключаецца ў тым, што аўтар артыкула ўпершыню аналізуе дыялагічны характар паэтычнага слова Э. Акуліна, які свядома ўводзіць у тэкст сваіх твораў інтэртэкстуальныя элементы. Ва ўводзінах абгрунтоўваецца выбар тэмы, апісваецца стан тэорыі па вывучаемым пытанні. Вызначаюцца асноўныя напрамкі ў вывучэнні інтэртэксту і інтэртэкстуальнасці ў паэтычнай творчасці Э. Акуліна. У асноўнай частцы аўтар ажыццяўляе аналіз інтэртэкстуальных элементаў у творчасці беларускага паэта. Сярод іх – алюзіі, рэмінісцэнцыі, цытаты, кампазіцыйныя сувязі тэкстаў. Аўтар артыкула звяртаецца да даследавання аўтарскай прыроды катэгорыі інтэртэкстуальнасці. У заключэнні аўтар прыходзіць да высновы, што інтэртэкстуальныя элементы ў паэзіі Э. Акуліна адрозніваюцца разнастайнасцю і прысутнічаюць на розных узроўнях мастацкага твора, пачынаючы ад кампазіцыйнага (архітэкстуальнасць), праз паратэкстуальныя адносіны (адносіны тэксту да сваёй пэўнай часткі), да непасрэдна інтэртэкстуальнасці.= The article is devoted to the analysis of intertextual elements in the poetry of E. Akulin. The novelty of the work is that the author of the article for the first time analyzes the dialogical nature of the poetic word of Eduard Akulin, who consciously introduces intertextual elements into the text of his works. The introduction justifies the choice of topics, describes the state of the theory on the issue under study. The main directions in the study of intertext and intertextuality in the poetic work of E. Akulin are determined. In the main part, the author analyzes intertextual elements in the work of the Belarusian poet. Among them are allusions, reminiscences, quotes, com-positional connections of texts. The author of the article turns to the study of the copyright nature of the category of intertextuality. In conclusion, the author concludes that the intertextual elements in the poetry of E. Akulin are diverse and present at different levels of a work of art, ranging from compositional (architextuality), through paratextual relations (the relationship of the text to its specific part), to directly intertextuality

LD-transpeptidases: the great unknown among the peptidoglycan cross-linkers

The peptidoglycan (PG) cell wall is an essential polymer for the shape and viability of bacteria. Its protective role is in great part provided by its mesh-like character. Therefore, PG-cross-linking enzymes like the penicillin-binding proteins (PBPs) are among the best targets for antibiotics. However, while PBPs have been in the spotlight for more than 50 years, another class of PG-cross-linking enzymes called LD-transpeptidases (LDTs) seemed to contribute less to PG synthesis and, thus, has kept an aura of mystery. In the last years, a number of studies have associated LDTs with cell wall adaptation to stress including β-lactam antibiotics, outer membrane stability, and toxin delivery, which has shed light onto the biological meaning of these proteins. Furthermore, as some species display a great abundance of LD-cross-links in their cell wall, it has been hypothesized that LDTs could also be the main synthetic PG-transpeptidases in some bacteria. In this review, we introduce these enzymes and their role in PG biosynthesis and we highlight the most recent advances in understanding their biological role in diverse species