Functions of the Essential Gene mraY in Cellular Morphogenesis and Development of the Filamentous Cyanobacterium Anabaena PCC 7120

Bacterial cell shape is determined by the peptidoglycan (PG) layer. The cyanobacterium Anabaena sp. PCC 7120 (Anabaena) is a filamentous strain with ovoid-shaped cells connected together with incomplete cell constriction. When deprived of combined nitrogen in the growth medium, about 5–10% of the cells differentiate into heterocysts, cells devoted to nitrogen fixation. It has been shown that PG synthesis is modulated during heterocyst development and some penicillin-binding proteins (PBPs) participating in PG synthesis are required for heterocyst morphogenesis or functioning. Anabaena has multiple PBPs with functional redundancy. In this study, in order to examine the function of PG synthesis and its relationship with heterocyst development, we created a conditional mutant of mraY, a gene necessary for the synthesis of the PG precursor, lipid I. We show that mraY is required for cell and filament integrity. Furthermore, when mraY expression was being limited, persistent septal PG synthetic activity was observed, resulting in increase in cell width. Under non-permissive conditions, filaments and cells were rapidly lysed, and no sign of heterocyst development within the time window allowed was detected after nitrogen starvation. When mraY expression was being limited, a high percentage of heterocyst doublets were found. These doublets are formed likely as a consequence of delayed cell division and persistent septal PG synthesis. MraY interacts with components of both the elongasome and the divisome, in particular those directly involved in PG synthesis, including HetF, which is required for both cell division and heterocyst formation.

Antibiotic resistance via bacterial cell shape-shifting

Bacteria have evolved to develop multiple strategies for antibiotic resistance by effectively reducing intracellular antibiotic concentrations or antibiotic binding affinities, but the role of cell morphology on antibiotic resistance remains poorly characterized. By analyzing cell morphological data of different bacterial species under antibiotic stress, we find that bacterial cells robustly reduce surface-to-volume ratio in response to most types of antibiotics. Using quantitative modelling we show that by reducing surface-to-volume ratio, bacteria can effectively reduce intracellular antibiotic concentration by decreasing antibiotic influx. The model predicts that bacteria can increase surface-to-volume ratio to promote antibiotic dilution if efflux pump activity is reduced, in agreement with data on membrane-transport inhibitors. Using the particular example of ribosome-targeting antibiotics, we present a systems-level model for the regulation of cell shape under antibiotic stress, and discuss feedback mechanisms that bacteria can harness to increase their fitness in the presence of antibiotics.

Cell morphology maintenance in Bacillus subtilis through balanced peptidoglycan synthesis and hydrolysis

The peptidoglycan layer is responsible for maintaining bacterial cell shape and permitting cell division. Cell wall growth is facilitated by peptidoglycan synthases and hydrolases and is potentially modulated by components of the central carbon metabolism. In Bacillus subtilis, UgtP synthesises the glucolipid precursor for lipoteichoic acid and has been suggested to function as a metabolic sensor governing cell size. Here we show that ugtP mutant cells have increased levels of cell wall precursors and changes in their peptidoglycan that suggest elevated dl-endopeptidase activity. The additional deletion of lytE, encoding a dl-endopeptidase important for cell elongation, in the ugtP mutant background produced cells with severe shape defects. Interestingly, the ugtP lytE mutant recovered normal rod-shape by acquiring mutations that decreased the expression of the peptidoglycan synthase PBP1. Together our results suggest that cells lacking ugtP must re-adjust the balance between peptidoglycan synthesis and hydrolysis to maintain proper cell morphology.

The evolution of spherical cell shape; progress and perspective

Bacterial cell shape is a key trait governing the extracellular and intracellular factors of bacterial life. Rod-like cell shape appears to be original which implies that the cell wall, division, and rod-like shape came together in ancient bacteria and that the myriad of shapes observed in extant bacteria have evolved from this ancestral shape. In order to understand its evolution, we must first understand how this trait is actively maintained through the construction and maintenance of the peptidoglycan cell wall. The proteins that are primarily responsible for cell shape are therefore the elements of the bacterial cytoskeleton, principally FtsZ, MreB, and the penicillin-binding proteins. MreB is particularly relevant in the transition between rod-like and spherical cell shape as it is often (but not always) lost early in the process. Here we will highlight what is known of this particular transition in cell shape and how it affects fitness before giving a brief perspective on what will be required in order to progress the field of cell shape evolution from a purely mechanistic discipline to one that has the perspective to both propose and to test reasonable hypotheses regarding the ecological drivers of cell shape change.

Sugar-Phosphate Metabolism Regulates Stationary-Phase Entry and Stalk Elongation in Caulobacter crescentus

ABSTRACT Bacteria have a variety of mechanisms for adapting to environmental perturbations. Changes in oxygen availability result in a switch between aerobic and anaerobic respiration, whereas iron limitation may lead to siderophore secretion. In addition to metabolic adaptations, many organisms respond by altering their cell shape. Caulobacter crescentus, when grown under phosphate-limiting conditions, dramatically elongates its polar stalk appendage. The stalk is hypothesized to facilitate phosphate uptake; however, the mechanistic details of stalk synthesis are not well characterized. We used a chemical mutagenesis approach to isolate and characterize stalk-deficient mutants, one of which had two mutations in the phosphomannose isomerase gene (manA) that were necessary and sufficient to inhibit stalk elongation. Transcription of the pho regulon was unaffected in the manA mutant; therefore, ManA plays a unique regulatory role in stalk synthesis. The mutant ManA had reduced enzymatic activity, resulting in a 5-fold increase in the intracellular fructose 6-phosphate/mannose 6-phosphate ratio. This metabolic imbalance impaired the synthesis of cellular envelope components derived from mannose 6-phosphate, namely, lipopolysaccharide O-antigen and exopolysaccharide. Furthermore, the manA mutations prevented C. crescentus cells from efficiently entering stationary phase. Deletion of the stationary-phase response regulator gene spdR inhibited stalk elongation in wild-type cells, while overproduction of the alarmone ppGpp, which triggers growth arrest and stationary-phase entry, increased stalk length in the manA mutant strain. These results demonstrate that sugar-phosphate metabolism regulates stalk elongation independently of phosphate starvation. IMPORTANCE Metabolic control of bacterial cell shape is an important mechanism for adapting to environmental perturbations. Caulobacter crescentus dramatically elongates its polar stalk appendage in response to phosphate starvation. To investigate the mechanism of this morphological adaptation, we isolated stalk-deficient mutants, one of which had mutations in the phosphomannose isomerase gene (manA) that blocked stalk elongation, despite normal activation of the phosphate starvation response. The mutant ManA resulted in an imbalance in sugar-phosphate concentrations, which had effects on the synthesis of cellular envelope components and entry into stationary phase. Due to the interconnectivity of metabolic pathways, our findings may suggest more generally that the modulation of bacterial cell shape involves the regulation of growth phase and the synthesis of cellular building blocks.

The core genome m5C methyltransferase JHP1050 (M.Hpy99III) plays an important role in orchestrating gene expression in Helicobacter pylori

ABSTRACTHelicobacter pylori encodes a large number of Restriction-Modification (R-M) systems despite its small genome.R-M systems have been described as “primitive immune systems” in bacteria, but the role of methylation in bacterial gene regulation and other processes is increasingly accepted. Every H.pylori strain harbours a unique set of R-M systems resulting in a highly diverse methylome. We identified a highly conserved GCGC-specific m5C MTase (JHP1050) that was predicted to be active in all of 459 H.pylori genome sequences analyzed. Transcriptome analysis of two H.pylori strains and their respective MTase mutants showed that inactivation of the MTase led to changes in the expression of 225 genes in strain J99, and 29 genes in strain BCM-300.10 genes were differentially expressed in both mutated strains. Combining bioinformatic analysis and site-directed mutagenesis, we demonstrated that motifs overlapping the promoter influence the expression of genes directly, while methylation of other motifs might cause secondary effects.Thus, m5C methylation modifies the transcription of multiple genes, affecting important phenotypic traits that include adherence to host cells, natural competence for DNA uptake, bacterial cell shape, and susceptibility to copper.

ENUMERASI DAN ANALISIS BAKTERI TANAH DI HUTAN LARANGAN ADAT RUMBIO (Enumeration And Bacteria Analysis of Soil on The Larangan Adat Rumbio Forest)

One of indicator of soil fertility is the population level of microbial in the soil. This research aims to determine the number of bacterial populations in the soil on yhe Larangan Adat Rumbi Forest with different levels of depth. This research has been carried out on January-February 2015 in the Laboratory of Pathology, Entomology and Microbiology, Faculty of Agriculture and Animal Sciences of State Islamic University of Sultan Syarif Kasim Riau. The method that was used namely observation method by taking soil samples on the Larangan Adat Rumbio Forest then calculated the number of colonies of bacteria and analyze the morphology of the bacteria that was obtained macroscopically and microscopically. Parameters measured were pH of soil, the population of bacteria, bacterial morphology, gram stain, and bacterial cell shape based on soil depth of 0-10 cm, 11-20 cm, 21-30 cm. Observations carried out two stages macroscopic and microscopic observation. The results showed the soil pH on the Larangan Adat Rumbio Forest was 4,11. Total population of bacteria at depth of 0-10 cm namely 3,0 x 109 CFU, then at a depth of 11-20 cm namely of 2,2 x 109 CFU and at depth of 21-30 cm namely 1,6 x 108 CFU. The Results of purification of culture was obtained six isolat two coccus and four bacil consisting of five gram negative bacteria and one gram positive. Need to do further research on bacteria identification to genus or species level.

A Single Dual-Function Enzyme Controls the Production of Inflammatory NOD Agonist Peptidoglycan Fragments by Neisseria gonorrhoeae

ABSTRACT Neisseria gonorrhoeae gonococcus (GC) is a Gram-negative betaproteobacterium and causative agent of the sexually transmitted infection gonorrhea. During growth, GC releases lipooligosaccharide (LOS) and peptidoglycan (PG) fragments, which contribute significantly to the inflammatory damage observed during human infection. In ascending infection of human Fallopian tubes, inflammation leads to increased risk of ectopic pregnancy, pelvic inflammatory disease, and sterility. Of the PG fragments released by GC, most are disaccharide peptide monomers, and of those, 80% have tripeptide stems despite the observation that tetrapeptide stems make up 80% of the assembled cell wall. We identified a serine-protease l,d-carboxypeptidase, NGO1274 (LdcA), as the enzyme responsible for converting cell wall tetrapeptide-stem PG to released tripeptide-stem PG. Unlike characterized cytoplasmic LdcA homologs in gammaproteobacteria, LdcA in GC is exported to the periplasm, and its localization is critical for its activity in modifying PG fragments for release. Distinct among other characterized l,d-carboxypeptidases, LdcA from GC is also capable of catalyzing the cleavage of specific peptide cross-bridges (endopeptidase activity). To define the role of ldcA in pathogenesis, we demonstrate that ldcA disruption results in both loss of NOD1-dependent NF-κB activation and decreased NOD2-dependent NF-κB activation while not affecting Toll-like receptor (TLR) agonist release. Since the human intracellular peptidoglycan receptor NOD1 (hNOD1) specifically recognizes PG fragments with a terminal meso-DAP rather than d-alanine, we conclude that LdcA is required for GC to provoke NOD1-dependent responses in cells of the human host. IMPORTANCE The macromolecular meshwork of peptidoglycan serves essential functions in determining bacterial cell shape, protecting against osmotic lysis, and defending cells from external assaults. The conserved peptidoglycan structure, however, is also recognized by eukaryotic pattern recognition receptors, which can trigger immune responses against bacteria. Many bacteria can induce an inflammatory response through the intracellular peptidoglycan receptor NOD1, but Neisseria gonorrhoeae serves as an extreme example, releasing fragments of peptidoglycan into the environment during growth that specifically antagonize human NOD1. Understanding the peptidoglycan breakdown mechanisms that allow Neisseria to promote NOD1 activation, rather than avoiding or suppressing immune detection, is critical to understanding the pathogenesis of this increasingly drug-resistant organism. We identify a peptidoglycan l,d-carboxypeptidase responsible for converting liberated peptidoglycan fragments into the human NOD1 agonist and find that the same enzyme has endopeptidase activity on certain peptidoglycan cross-links, the first described combination of those two activities in a single enzyme. IMPORTANCE The macromolecular meshwork of peptidoglycan serves essential functions in determining bacterial cell shape, protecting against osmotic lysis, and defending cells from external assaults. The conserved peptidoglycan structure, however, is also recognized by eukaryotic pattern recognition receptors, which can trigger immune responses against bacteria. Many bacteria can induce an inflammatory response through the intracellular peptidoglycan receptor NOD1, but Neisseria gonorrhoeae serves as an extreme example, releasing fragments of peptidoglycan into the environment during growth that specifically antagonize human NOD1. Understanding the peptidoglycan breakdown mechanisms that allow Neisseria to promote NOD1 activation, rather than avoiding or suppressing immune detection, is critical to understanding the pathogenesis of this increasingly drug-resistant organism. We identify a peptidoglycan l,d-carboxypeptidase responsible for converting liberated peptidoglycan fragments into the human NOD1 agonist and find that the same enzyme has endopeptidase activity on certain peptidoglycan cross-links, the first described combination of those two activities in a single enzyme.