Kocuria rosea - an overview (2023)

RNS Production and Interaction with Prokaryotes

The inhibitory effect of NO on E. coli was first noted in 1965 (Russell, 1965). Subsequent studies nearly 20 years later expanded the list of NO-sensitive species to include Micrococcus luteus, Micrococcus roseus and Staphylococcus aureus, but opportunistic pathogen Serratia marcescens and several Bacillus species were found to be NO resistant (Mancinelli and McKay, 1983). The NO tolerance displayed by Bacillus is consistent with its denitrification activity (Mevel and Prieur, 2000). In addition to the RNS produced by denitrification, microbes encounter RNS that are produced by a host’s immune system or by nitrite acidification, with the latter two sources being the most relevant to human health.

The mammalian immune system generates micromolar levels of NO (Krieglstein et al., 2001), a concentration that is often sufficient to inhibit microbial growth. NO is enzymatically produced from L-arginine by a variety of nitric oxide synthases (NOS) in plants and mammals, with the mammalian inducible NOS (iNOS) being the most significant enzymatic source of NO for pathogens. While some plants employ an iNOS in their immune arsenal, NO appears to be used as a signal instead of a weapon (Delledonne et al., 1998). NO is produced as an intermediate species during the reduction of nitrate to nitrite (Remde and Conrad, 1991a). During nitrite respiration, microbes are exposed to nanomolar levels of NO generated by nitrite reductase (Goretski et al., 1990; Remde and Conrad, 1991b). Although this low level of NO does not appear to be bacteriostatic, the observed activation of NO detoxification systems by these low levels of NO (Corker and Poole, 2003) implies that the NO-mediated damage is sufficient to justify the cost of NO detoxification. Similarly, nitrite can be reduced to NO and NO+ in highly acidic environments, such as the gastric juices. Organisms attempting to persist/survive in this type of environment, such as Helicobacter pylori (Dykhuizen et al., 1998; Thiele et al., 2005), must be able to cope with the micromolar concentrations of NO produced (McKnight et al., 1997).

One of the hallmarks of NO chemistry is its reactivity with metal groups (Wink et al., 1999). For bacterial species attempting to grow in the presence of NO, this translates to damage of metal-containing proteins. These protein-associated metal groups include, but are not limited to, iron–sulfur [Fe-S] clusters and heme groups. As reviewed in Johnson et al. (2005), [Fe-S]-containing proteins play key roles in metabolic and regulatory processes ranging from amino acid (AA) biosynthesis to the superoxide stress response. Unfortunately, the very properties that underlie their catalytic properties also make them targets for damage by compounds such as RNS and reactive oxygen species (ROS). Still, the sensitivity of these clusters to RNS- and ROS-mediated damage is variable. While E. coli’s branched chain amino acid (BCAA) biosynthesis protein dihydroxyacid dehydratase is particularly sensitive to damage from NO (Hyduke et al., 2007) and superoxide (Flint et al., 1993), other Fe-S-containing enzymes, such as the spinach dihydroxyacid dehydratase, are robust to damage from superoxide (Flint et al., 1993). Eukaryotic soluble guanylate cyclase (sGC) is the most well characterized heme-containing NO target, and a prokaryotic counterpart with femtomolar sensitivity to NO was found in Clostridium botulinum, the causative organism of botulism (Nioche et al., 2004). Bacterial species often exploit the vulnerability of these metal groups and use them as sensors, such as the Fe-S cluster in NsrR (Yuki et al., 2008), the non-heme iron center of NorR (D’Autreaux et al., 2005), and the heme group in Paracoccus denitrificans NO reductase regulator NNR (Lee et al., 2006).

As opposed to the metal reactivity of NO, nitrosothiols are noted for their reactivity with thiol groups (Hogg, 2002). This reactivity can inhibit bacterial growth by damaging enzymatic cysteine residues (Wink et al., 1994) or by reacting with and depleting free cysteine or homocysteine (Jarboe et al., 2008).

Taxonomic Status of Micrococcus and Micrococcaceae

Bacteria belonging to the genus Micrococcus are Gram-positive, spherical, 0.5–2.0 μm in diameter, nonsporing, and seldom motile. They occur in pairs, tetrads, or irregular clusters, not in chains. They are strictly aerobic, chemoorganotrophs, with a respiratory metabolism, and generally produce little or no acid from carbohydrates. They are catalase positive and often oxidase positive, although weakly. They contain cytochromes and are resistant to lysostaphin. They form colonies usually pigmented in shades of yellow or red. They grow on simple media and are usually halotolerant, able to grow with 5% NaCl. Their optimum temperature generally ranges from 25 to 37 °C. Their G+C content of DNA ranges from 64 to 75 mol%.

Micrococcus traditionally has been included in the Micrococcaceae family, together with the genera Staphylococcus, Stomatococcus, and Planococcus of aerobic and facultative anaerobic Gram-positive, catalase-positive cocci. However, a higher 5S rRNA sequence similarity of Micrococcus luteus strains ATCC 9341 and ATCC 4698 with Streptomyces griseus 45-H than with Staphylococcus epidermidis strain ATCC 14990 and Staphylococcus aureus strain Smith was found. According to some authors, Streptomyces and Micrococcus, characterized by a high genomic G+C content, emerged from the Gram-positive bacterial stem at an early time during bacterial evolution, and their unique 5S rRNA secondary structure is closer to the Gram-negative type than to the Gram-positive type.

A new genus, Macrococcus, was proposed in 1998 within the Micrococcaceae family on the basis of a phylogenetic analysis comparing 16S rRNA sequences, DNA–DNA liquid hybridization, DNA base composition, normalized ribotype patterns, macrorestriction pattern analysis, and estimation of genome size using pulsed field gel electrophoresis (PFGE) cell wall composition, phenotypic characteristics, and plasmid profiles. Compared with members of the genus Staphylococcus, their closest relatives, Macrococcus showed lower 16S rRNA sequence similarities (93.4–95.3%), higher G+C content in the DNA (38–45 mol%), absence of cell wall teichoic acids, with the possible exception of Micrococcus caseolyticus, unique ribotype pattern types and macrorestriction patterns, smaller genome size, of approximately 1500–1800 kb, and generally larger Gram-stained cell size, of 1.1–2.5 μm in diameter. The new genus is integrated by the four species M. caseolyticus (formerly Staphylococcus caseolyticus), Micrococcus bovicus, Micrococcus equipercicus, and Micrococcus carouselicus.

Nine species of Micrococcus, Micrococcus agilis, Micrococcus halobius, Micrococcus kristinae, M. luteus, Micrococcus lylae, Micrococcus nishinomiyaensis, Micrococcus roseus, Micrococcus sedentarius, and Micrococcus varians were recognized until 1995. Data on the G+C content of the DNA, fatty and mycolic acid patterns, peptidoglycan type, and 16S rDNA sequences revealed large heterogeneity within this genus. Consequently, strains previously identified as belonging to seven species of Micrococcus were transferred to genera Arthrobacter, Dermacoccus gen. nov., Kocuria gen. nov., Kytococcus gen. nov., and Nesterenkonia gen. nov. Thus, M. agilis, which grows best at 22–25 °C, has been renamed as Arthrobacter agilis; M. nishinomiyaensis as Dermacoccus nishinomiyaensis; M. kristinae, M. roseus, and M. varians as Kocuria kristinae, Kocuria rosea, and Kocuria varians, respectively; M. sedentarius as Kytococcus sedentarius; and M. halobius, which requires 5% NaCl in the culture medium, as Nesterenkonia halobia. Currently, only two species, M. luteus and M. lylae, remain in the genus Micrococcus.

After these changes, a new description of the genus Micrococcus (Cohn, 1872; Stackebrandt et al., 1995) was given, as follows: Micrococcus cells are spherical and nonmotile; endospores are nonformed, Gram-positive, and aerobic; chemoorganotrophic; metabolism is strictly respiratory; catalase and oxidase positive; and mesophilic and nonhalophilic. The peptidoglycan contains l-lysine as the diagnostic amino acid. The peptidoglycan variation is either A2, with the interpeptide bridge consisting of a peptide subunit, or A4α. The predominant menaquinones are either MK-8 and MK-8(H₂) or MK-8(H₂); MK-7 or MK-7(H₂) and MK-9(H₂) occur in minor amounts. The cytochromes are cytochromes aa₃, b₅₅₇, b₅₆₇, and d₆₂₆; cytochromes c₅₅₀, c₅₅₁, b₅₆₃, b₅₆₄, and b₅₆₇ may be present. Mycolic acids and teichoic acids are absent; teichuronic acids may be present. Mannosamine-uronic acid may be present as an amino sugar in the cell wall polysaccharide. Cellular fatty acids are iso- and anteiso-branched fatty acids, with anteiso-C_15:0 and iso-C_15:0 predominating. Polar lipids are phosphatidylglycerol, diphosphatidylglycerol, and unknown ninhydrin-negative phospholipids and glycolipids; phosphatidylinositol may be present. The major aliphatic hydrocarbons (br-Δ-C) are C₂₇ to C₂₉ hydrocarbons. The G+C content of the DNA is 69–76 mol% (as determined by the T_m method). The primary habitat is mammalian skin. The type species is M. luteus (Schroeter; Cohn, 1872).

Emended descriptions of the genus Micrococcus and the species M. luteus and M. lylae were given in 2002, on the basis of a polyphasic approach to the classification of nine yellow-pigmented, spherical bacterial strains isolated from various sources. In addition to the properties given in the 1995 genus description, members of the genus Micrococcus show several common characteristics. Growth occurs up to pH 10. The polar lipids are phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, an unknown glycolipid, and an unknown ninhydrin-negative phospholipid. l-arabinose, p-arbutin, d-cellobiose, d-galactose, d-melibiose, d-ribose, and salicin are not assimilated. Members of the genus share the Micrococcaceae-specific signature nucleotides at positions 293–304, 610, 598, 615–625, 1025–1036, 1026–1035, 1265–1270, and 1278 of the 16S rRNA gene sequence (Escherichia coli numbering) and lack the signature nucleotides at positions 640, 839–847, and 859. The description of M. luteus and M. lylae also was emended, and M. luteus was dissected into three biovars, distinguishable by chemotaxonomic and biochemical traits: biovar I, represented by M. luteus DSM 20030^T; biovar II, represented by M. luteus DSM 14234, and biovar III, represented by M. luteus DSM 14235.

In spite of these taxonomic studies, however, the nine species M. agilis, M. halobius, M. kristinae, M. luteus, M. lylae, M. nishinomiyaensis, M. roseus, M. sedentarius, and M. varians were still recognized in 2006 in the handbook The Prokaryotes, which included a scheme for the differentiation of these species based on physiological and chemotaxonomic characteristics.

A The microflora of spines versus photosynthetic leaf parts

To understand whether spines are a different habitat than green photosynthetic leaf surfaces when it comes to the microbial flora, we compared the microbial communities of the phyllosphere on spines to the photosynthetic leaf surfaces of the palm tree W. filifera (unpublished study). Cotton palm trees have a single straight stem that can reach more than 20m in height and are usually 40–70cm in diameter (Fig. 4.2A). The petioles, which may reach more than 1m in length, usually have two rows of sharp spines along their right and left margins (Fig. 4.2C). Every cotton palm tree carries thousands of colorful aposematic spines. The spines may be straight, curved, or recurved and are yellow, orange, or brown in color depending on leaf age and individual tree characteristics, and a panel of the same color connects them along the edge of the petioles, enhancing spine conspicuousness.

Spines and green photosynthetic leaf surfaces (Fig. 4.1B and C) were sampled from different trees in Northern Israel in December 2008, March 2009, May 2009, and June 2009 (unpublished). Using culturable methods, we isolated and identified a total of 97 bacterial isolates (accession numbers HM163472–HM163568), which were then classified as pathogens or nonpathogens according to the scientific literature records. Fifteen pathogenic species were identified from spines, and 18 pathogenic species were identified from the green photosynthetic leaf surfaces (Table 4.1, columns 5 and 6). The following pathogenic or opportunistic pathogenic bacteria—Pantoea brenneri, Pseudomonas stutzeri, Acinetobacter johnsonii, Acinetobacter lwoffii, Clostridium sordellii, Bacillus anthracis, Staphylococcus hominis subsp. hominis, and Kocuria rosea—were isolated only from W. filifera spines and were not identified on green photosynthetic leaf surfaces (Table 4.1). In sum (including the nonpathogenic species), 28 bacterial species were found on the spines and 36 species on the green photosynthetic leaf surfaces. Out of these, 11 species were found on both the spines and green photosynthetic leaf surfaces. This data was used to calculate Sorensen's relatedness index (values are between 0 and 1.0; Wolda, 1981). The similarity between the bacterial flora on green photosynthetic leaf surfaces and spines according to Sorensen's index was 0.343, indicating that the microflora of the spines in cotton palms is different from that of the green photosynthetic leaf surfaces. Thus, the microbe–spine combination seems to be an important factor in the common evolution of the aposematic coloration of spiny, thorny, and prickly plants. Hence, bacteria that inhabit spines or thorns in plants seem to have enhanced the common, convergent evolution of aposematism in these organisms.

6 Biological activities

Several biological assays have been carried out to support the ethnomedicinal claims of Homalanthus species. The biological assays conducted were in vitro, together with three in vivo assays focusing on H. acuminatus, H. nervosus, H. nutans, H. novoguineensis, and H. populneus. Among the Homalanthus species, H. nutans and H. nervosus are the most explored ones. Table 4 summarizes the biological activities of the genus.